Dendrimers via Click Reaction To Enhance Drug ... - ACS Publications

Research Article Cite This: ACS Appl. Mater. Interfaces 2017, 9, 42459−42470

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MMP-2-Sensitive HA End-Conjugated Poly(amidoamine) Dendrimers via Click Reaction To Enhance Drug Penetration into Solid Tumor Min Han,†,§ Ming-Yi Huang-Fu,†,§ Wang-Wei Guo,† Ning-Ning Guo,† JieJian Chen,‡ Hui-Na Liu,† Zhi-Qi Xie,† Meng-Ting Lin,† Qi-Chun Wei,‡ and Jian-Qing Gao*,† †

College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, P. R. China Department of Radiation Oncology, Key Laboratory of Cancer Prevention and Intervention, the Second Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310058, China

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ABSTRACT: Currently, the limited penetration of nanoparticles remains a major challenge for antitumor nanomedicine to penetrate into the tumor tissues. Herein, we propose a size-shrinkable drug delivery system based on a polysaccharide-modified dendrimer with tumor microenvironment responsiveness for the first time to our knowledge, which was formed by conjugating the terminal glucose of hyaluronic acid (HA) to the superficial amidogen of poly(amidoamine) (PAMAM), using a matrix metalloproteinase-2 (MMP-2)cleavable peptide (PLGLAG) via click reaction. These nanoparticles had an initial size of ∼200 nm, but once deposited in the presence of MMP-2, they experienced a dramatic and fast size change and dissociated into their dendrimer building blocks (∼10 nm in diameter) because of cleavage of PLGLAG. This rapid size-shrinking characteristic not only promoted nanoparticle extravasation and accumulation in tumors benefited from the enhanced permeability and retention effect but also achieved faster nanoparticle diffusion and penetration. We have further conducted comparative studies of MMP-2sensitive macromolecules (HA-pep-PAMAM) and MMP-2-insensitive macromolecules (HA−PAMAM) synthesized with a similar particle size, surface charge, and chemical composition and evaluated in both monolayer cells and multicellular spheroids. The results confirmed that the enzyme-responsive size shrink is an implementable strategy to enhance drug penetration and to improve therapeutic efficacy. Meanwhile, macromolecule-based nanoparticles with size-variable characteristics not only promote drug penetration, but they can also be used as gene delivery systems, suggesting great potential as nano-delivery systems. KEYWORDS: nanoparticle, tumor penetration, particle size, tumor microenvironment, enzyme-responsive

1. INTRODUCTION

therapeutic drugs are significantly blocked from entering adjacent areas of the tumor vasculature after extravasation from the tumor vessels. This is caused by high interstitial fluid pressure (IFP) and intensive extracellular matrix (ECM), and it greatly reduces the therapeutic effect of the nanoparticles.4 Addressing the dilemma of improving the efficacy of treatment and refining the physical and chemical properties of nanoparticles, such as size and shape, provides a solution to these problems. It has been demonstrated that larger nanoparticles (∼50−200 nm) are usually maintained at high levels across the tumor vasculature and accumulate around the blood vessels,5,6 benefitting the EPR effect. While larger nanoparticles penetrate and distribute in a solid tumor poorly, smaller nanoparticles are favorable for tumor penetration, benefiting from a reduced diffusion barrier, but they usually are

Cancer is one of the most serious health threats for humans, which has aroused great concern.1 Currently, chemotherapy is considered an effective approach for treating human cancers,2 but the existence of systemic toxicity, lack of tumor targeting, and tumor multidrug resistance limits its wide application in clinical practice. In addition, the limited tumor penetration of nanoparticles remains one of the major challenges of cancer nanomedicine. The study about sophisticated nanoparticles for the targeted drug delivery to solid tumors has led to great expectations to achieve better treatment efficacy and minimize systemic toxicity. To obtain effective tumor therapy, drugs must penetrate tumor tissue deeply to target as many cancer cells as possible. Compared with that of small-molecule drugs, nanoparticles have a relatively large size, which allows them to effectively accumulate in solid tumors through the enhanced permeability and retention (EPR) effect.3 However, their large size seems to be a hindrance to the deep penetration of nanoparticles to the tumor. It has been shown that nanoscale © 2017 American Chemical Society

Received: July 12, 2017 Accepted: November 16, 2017 Published: November 16, 2017 42459

DOI: 10.1021/acsami.7b10098 ACS Appl. Mater. Interfaces 2017, 9, 42459−42470

Research Article

ACS Applied Materials & Interfaces

Figure 1. Schematic illustration to show the shrinkage of the HA-pep-PAMAM from 200 to 10 nm triggered by MMP-2, a protease highly expressed in the tumor extracellular matrix, thus enabling penetration into deep tumor tissue.

This approach supports reductive amination reactions and ensures that each HA can only be connected with PAMAM in a controlled manner, by avoiding the participation of the carboxylic acid groups in HA. The physicochemical properties and MMP-2 sensitivity of the resulting HA-pep-PAMAM/DOX nanoparticles were studied. Then, the uptake in cell monolayers, penetration into tumor spheroids, and anticancer activity in tumor-bearing nude mice were investigated for HApep-PAMAM/DOX using A549 and MCF-7 cells. A schematic diagram (Figure 1) shows the three-step strategy for improving the penetration of drugs into the tumor site by the introduction of a cleavable peptide to the nanoparticles.

cleared quickly from the body. The design and development of size-shrinkable drug delivery systems that maintain a large initial particle size to extend the blood circulation time but that degrade into a smaller size in the tumor to achieve a deeper penetration and effective distribution has surmounted the dilemma.7−9 Typical systems have emerged, such as mesoporous silica or Au nanoparticles, with smaller particle sizes.10,11 However, these materials employed that are designed to achieve better penetration and antitumor efficacy may have poor biodegradability.12 Compared with other poly(amidoamine) (PAMAM) dendrimers, we first designed a size-shrinkable drug delivery system responsive to the tumor microenvironment based on a polysaccharide-modified dendrimer in the present study,13,14 which contains PAMAM, hyaluronic acid (HA), and MMP-2sensitive peptide linker (PLGLAG), using doxorubicin (DOX) as a model drug. PAMAM is one of the most widely studied dendrimers.15,16 However, because of its high charge, the inherent toxicity of PAMAM dendrimers hinders its application. In addition, as a linear polysaccharide with repeating disaccharide units, HA is widely distributed in the human body and has been used in nanoparticle-related research recently because of its biodegradability. HA also has a strong affinity for CD44 receptors that are overexpressed on many cancer cells and cancer stem cells.17 Instead of inorganic or other synthetic polymers, HA-based nanoparticles revealed favorable biodegradability and physiological stability, prolonged circulation in vivo, and increased accumulation in tumor sites. As for the conjunction between HA and PAMAM, the position of the linker is considerably important. Modification of the abundant amino groups on PAMAM is a common means for the synthesis of nanoparticles, but the binding site of HA, which has many carboxyl groups, to PAMAM is difficult to control. Despite cross-linking between multi-amino and multi-carboxyl groups enhancing the structural stability of the nanoparticles in the blood circulation, it could inhibit the ability of the enzymes at the target site to release the drug, thus reducing therapeutic efficacy. Furthermore, excess modification of the carboxylic acid groups in the HA structure would compromise its targetability. In this study, we used end-activated HA in the reaction to avoid the participation of the many −COOH groups. The terminal end of HA, which can be converted to an aldehyde group after a ring-opening reaction, is used to react with an acceptor amino group to achieve a 1:1 stoichiometric ratio.

2. EXPERIMENTAL SECTION 2.1. Materials and Animals. DOX hydrochloride was purchased from Zhejiang Hisun Pharmaceutical Co. Ltd. (Zhejiang, China), and HA (MW = 17, 48 kDa) was provided by Shandong Freda Biopharm Co. Ltd. (Shandong, China). The MMP-2 protease-sensitive peptide substrate Gly-PLGLAG-Cys was synthesized by GL Biotech Co., Ltd. (Shanghai, China). The PAMAM dendrimer (ethylenediamine core, G5.0) was purchased from Weihai CY Dendrimer Technology Co., Ltd. (Weihai, China). 1-(2-Aminoethyl)maleimide (AEM) and hexadecyl trimethyl ammonium bromide (CTAB) were purchased from Sigma-Aldrich Co. (USA). Sodium triacetoxyborohydride (STAB) was purchased from Tokyo Chemical Industry (Japan). Agarose with a low melting point was purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM) was provided by Hyclone (Logan, UT, USA), trypsin− ethylenediaminetetraacetic acid (EDTA) solution (0.5% trypsin, 5.3 mM EDTA tetrasodium) was purchased from Jinuo Biomedical Technology Co., Ltd. (Hangzhou, China), and fetal bovine serum (FBS) was provided by Thermo Fisher Scientific (Waltham, MA, USA). CCK8-assay kit were purchased from Beyotime Biotechnology Co. (Jiangsu, China). N-Hydroxysuccinimide (NHS) was purchased from Aladdin Co. (Shanghai, China). N-(3-Dimethyl amino propyl)N′-ethylcarbodiimide hydrochloride (EDC) was purchased from Sigma-Aldrich (USA). The anti-CD31 antibody was purchased from HangZhou HuaAn Biotechnology Co. Ltd. (Hangzhou, China). Distilled water used in the synthesis was prepared using a Milli-Q (Millipore, USA). All other chemicals used in the study were of analytical grade. The human non-small cell lung cancer (A549) cells and breast cancer (MCF-7) cells [Institute of Biochemistry and Cell Biology of Chinese Academy of Science (Shanghai, China)] were grown in complete growth media (DMEM supplemented with 1× PS and 10% FBS) at 37 °C in 5% CO2. BALB/c nude mice (18 ± 2 g, female) were purchased from SLACCAS (Shanghai, China). The animals were maintained under 42460


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3 × 103 cells/well in 96-well plates. Cells were incubated with DOX· HCl solution, PAMAM/DOX, HA−PAMAM/DOX, HA-pepPAMAM/DOX, or MMP-2-treated HA-pep-PAMAM/DOX at various drug concentrations (0.0625−2 μg/mL) for 48 h followed by the CCK-8 assay. Briefly, 15 μL of the CCK-8 reagent was added to 100 μL fresh culture medium per well and incubated with the cells at 37 °C for 2 h. To calculate the half-maximal inhibitory concentration (IC50) value of each group, the fluorescence intensity was measured on a microplate reader at λex = 560 nm and λem = 590 nm. The toxicities of HA−PAMAM and HA-pep-PAMAM were also assessed in the MCF-7 and A549 cells by the same protocol. 2.8. Tumor Spheroids Assay Penetration. It has been demonstrated that monolayer cell cultures cannot fully represent in vivo tumors owing to their accountable differences in cellular heterogeneity, nutrient and oxygen gradients, cell−cell interactions, matrix deposition, and gene expression profiles, resulting in different drug responses and poor in vitro−in vivo correlations.23 Compared to that of monolayer cultures, tumor spheroids provide better target cells and are in vitro models that are more representative of real tumor conditions.24 A549 spheroids were produced by the liquid overlay method as previously described.20,25 Briefly, cells were separated from monolayers, and single-cell suspension (400 μL per well, containing 1 × 104 cells) was transferred into flat-bottomed 48-well plates precoated with 2% agarose. The plates were then centrifuged at 100 rpm for 10 min, and the cells were incubated for approximately 4 days as described above for monolayer cells, except that the cell culture medium was partially replaced with fresh medium (200 μL) every other day. Tumor spheroids of A549 cells were used to evaluate the tumor-penetrating ability of DOX formulations. Sterile agarose solution (2%, w/v) was added to each well of the 48-well plate. After the solidification of the agarose solution, A549 cells (3500 cells per well) were seeded in the plate. The cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2. After 4 days, the formed tumor spheroids were incubated with DOX, PAMAM/DOX, HA-pepPAMAM/DOX, HA−PAMAM/DOX, or HA-pep-PAMAM/DOX pretreated with MMP-2 for 4 h. After culturing for 4 h, the tumor spheroids obtained were washed with cold PBS 3 times. The tumor spheroids were then fixed with 4% (w/v) paraformaldehyde for 15 min. The penetration depth was observed by confocal laser scanning microscopy (CLSM). 2.9. In Vitro Antitumor Spheroids Effect. A549 tumor spheroids were established as mentioned above. When their diameters reached ∼400 μm, the tumor spheroids were divided into four groups (n = 6 per group) and were treated with DOX, PAMAM/DOX, HA− PAMAM/DOX, or HA-pep-PAMAM/DOX with or without MMP-2 pretreatment (final DOX concentration, 4 μg/mL). The tumor spheroid size was measured and morphology was recorded every day during the study. The tumor spheroid volume was calculated by the formula: V = (width2 × length)/2. 2.10. Biodistribution. To investigate the real-time distribution and tumor accumulation ability of the conjugate, indocyanine green (ICG), an amphiphilic carbocyanine dye that strongly absorbs and fluoresces in the near-infrared (NIR) region and is used often in diagnostic and therapeutic applications, was encapsulated in the nanoparticles.26−28 In the study, nude female mice (4 weeks old) received a subcutaneous injection of 0.1 mL of A549 cancer cell suspension containing 3.5 × 106 cells, as previously described with some appropriate modification.29 A549 tumor-bearing nude mice were intravenously injected with free ICG, PAMAM/ICG HA−PAMAM/ ICG, or HA-pep-PAMAM/ICG individually (ICG concentration, 2 mg/kg). At 2, 6, 12, and 48 h after injection, the biodistribution of the fluorescent particles was observed in vivo. At 48 h, the mice were sacrificed, and the tumor and organs (heart, liver, spleen, lung, and kidney) were harvested and then imaged using the Maestro in vivo imaging system (Cambridge Research and Instrumentation, USA). 2.11. Antitumor Effect. A549 tumor-bearing mice were established as described above. One week after implantation, the mice were randomly divided into six groups (six mice per group): PBS, DOX solution, PAMAM/DOX, HA-pep-PAMAM/DOX, HA− PAMAM/DOX, and HA-pep-PAMAM. Each A549 tumor-bearing

standard housing conditions, and all related experiments were conducted according to guidelines which have been evaluated and approved by the ethics committee of Zhejiang University. 2.2. Synthesis of HA-pep-PAMAM. HA (51.0 mg) was dissolved in 5 mL of 0.1 M borate buffer (pH 8.5, 0.3 mol/L sodium chloride). Then, 30.5 mg of AEM and 25.4 mg of STAB were added to the HA solution and stirred at 60 °C for 4 days in a water bath. Subsequently, the product solution was dialyzed against water to remove unreacted substances. The purified HA−AEM was lyophilized and stored at −20 °C. PAMAM G5.0 (28.0 mg) was dissolved in distilled water (2.0 mg/ mL). Peptide (6.9 mg) was dissolved in double-distilled water. After the addition of catalyzer EDC and NHS, the solution was added to the prepared PAMAM solution. The mixture was adjusted to pH 6.0 and reacted at room temperature (RT) for 4 h. The product solution was dialyzed against water to remove unreacted substances. The purified PAMAM-pep was lyophilized and stored at −20 °C. The above-mentioned products were mixed in a 1:1 mass ratio and stirred for 4 h at 0.2 M phosphate-buffered saline (PBS) (pH 7.4) at RT, and then they were dialyzed for 48 h against PBS to give HA-pepPAMAM. 2.3. Synthesis of HA−PAMAM. PAMAM G5.0 (28.8 mg) was added to 25 mL of 0.1 M borate buffer (pH 8.5, 0.3 M NaCl). HA (51.0 mg) was added and stirred until the HA was completely dissolved. Then, NaBH3CN (25.4 mg) was added to the reaction solution and then stirred at 60 °C for 4 days in a water bath. Subsequently, the product solution was dialyzed against PBS to remove unreacted substances. The purified HA−PAMAM was stored at 4 °C. 2.4. Cleavage of HA-pep-PAMAM by MMP-2. HA-pepPAMAM nanoparticles (4 mg/mL) were incubated at 37 °C with 0.5 mg/mL MMP-2 in 25 mM HEPES buffers (pH 7.4) including 5 mM CaCl2. After digestion by the enzyme, the mixture was centrifuged at 20 000 rpm for 15 min to remove the precipitates. The HA that released from enzymatic reaction existed in the supernatants and was detected by the CTAB method.18,19 In addition, to investigate the MMP-2 protease-responsive property of HA-pep-PAMAM, the morphology and the size changes of the nanoparticles were detected after incubation with the MMP-2 protease for 6 h. 2.5. Nanoparticle Characterization. The morphology of HApep-PAMAM/DOX (with/without MMP-2 pretreatment) and HA− PAMAM/DOX was captured by a transmission electron microscope (JEM-1230, JEOL, Japan). The zeta potential and particle size were analyzed by dynamic light scanning analysis using a Zetasizer Nano ZS instrument (Malvern, UK). The in vitro DOX release from different formulations was evaluated using a micro-plate spectrophotometer (Synergy MX M5, Molecular Devices, USA). The HA-pep-PAMAM/ DOX or HA−PAMAM/DOX was resuspended in PBS at pH 7.4. Meanwhile, free DOX was dispersed in PBS at pH 7.4 as the control. The fluorescence emission spectra of HA-pep-PAMAM/DOX were obtained at different time sampling intervals from 0 to 96 h. All procedures were carried out in the dark. 2.6. Cellular Uptake Study. For the cellular uptake study, the cells were seeded in six-well plates at a cell density of 1.0 × 105 cells/ well. After overnight incubation, the cells were washed by pH 7.4 PBS and then were incubated with DOX solution or HA-pep-PAMAM/ DOX or HA−PAMAM/DOX in 1 mL/well of media for 4 h at 37 °C. To study the influence of MMP-2, HA-pep-PAMAM/DOX was pretreated with human MMP-2 for 4 h before incubating with the cells. After incubating the cells for another 4 h, the cells were analyzed by flow cytometry. Briefly, the cells were washed with cold PBS, trypsinized and collected by centrifugation at 1200 rpm for 5 min, resuspended in 0.5 mL PBS, and followed by intensity analysis on a flow cytometer (Accuri C6 flow cytometer, BD Biosciences). The data were collected and analyzed using Accuri CFlow Plus software (10 000 cell counts per sample). The cells were gated upon acquisition using forward scatter versus side scatter to eliminate dead cells and debris. 2.7. Cell Viability Assay. To evaluate the anticancer efficacy of HA-pep-PAMAM/DOX, A549 (high MMP-2 expression cells)20 and MCF-7 cells without MMP-2 overexpression21,22 were grown at about 42461


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Figure 2. Schematic showing the synthesis of HA-pep-PAMAM (A) and HA−PAMAM (B). (i) Sodium triacetoxyborohydride, 0.1 M borate buffer including 0.3 M sodium chloride, pH 8.5, 60 °C, 96 h; (ii) NHS, EDC, pH 6.0, RT, 4 h; (iii) 0.2 M PBS (pH 7.4), RT, 4 h; (iv) sodium cyanoborohydride, 0.1 M borate buffer including 0.3 M sodium chloride, pH 8.5, 60 °C, 96 h. mouse was injected intravenously at a dose of 2 mg/kg DOX every 3 days for 6 times. The tumor volume and body weight were measured before every injection, and the tumor volume was calculated as the volume = (width2 × length)/2. At the end of treatment, blood samples were obtained from the ocular vein. To obtain the serum, the whole blood sample was centrifuged at 3000 rpm for 15 min. To assess the liver function, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in serum were measured. cardiotoxicity was determined based on lactic dehydrogenase (LDH) and creatine kinase-MB (CK-MB) levels. These biochemical parameters were measured by an automated biochemical analyzer. Mice were sacrificed at the end of the study, and the organs were extracted. A small piece of the heart, liver, spleen, lung, kidney, and tumor were fixed in 4% paraformaldehyde, embedded in paraffin, sliced into 5 μm thicknesses, and then placed onto glass slides. The organs and tumor sections were stained with

hematoxylin and eosin (H & E) and were estimated by light microscopy for a histological study of toxicity. Additionally, sections of the heart, liver, and tumors were stained by Hoechst 33342, and tumor cell apoptosis was analyzed. 2.12. Penetration Assay. To evaluate the permeability of nanoparticles, Cy5.5-labeled HA-pep-PAMAM was intravenously injected into the A549 tumor-bearing mice. Mice were sacrificed 24 h after administration, and the harvested tumors were frozen and cut into 16 μm slices for histological analysis. Then, sections were fixed in 4% paraformaldehyde for 10 min and blocked with 10% bovine serum albumin for 1 h, followed by incubation with a rat anti-CD31 antibody (1:50) overnight at 4 °C. Tumor slices were washed 3 times with PBS and incubated with a fluorescein isothiocyanate-conjugated goat antirat immunoglobulin G secondary antibody (1:100) and 4′,6-diamidino-2phenylindole for 3 h and then rinsed and sealed with a coverslip. The 42462


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Figure 3. Characterization of HA-pep-PAMAM. (A) TEM image of HA-pep-PAMAM before incubation with MMP-2. (B) TEM image of HA-pepPAMAM after incubation with MMP-2 for 4 h. (C) The particle size of HA-pep-PAMAM before incubation with MMP-2. (D) The size change of HA-pep-PAMAM after incubation with 0.5 mg/mLMMP-2 for 4 h at 37 °C. (E) UV−vis detection of HA removed from HA-pep-PAMAM with 0.5 mg/mL MMP-2 at different temperatures.

absorption peaks at 3094 cm−1, representing the stretching vibration of NH of the amino groups in PAMAM, which was not shown in HA because of the lack of amino groups. This vibration signal was weaker in HA-pep-PAMAM and HA− PAMAM than in PAMAM, indicating that HA was introduced and reacted with the amino groups on the surface of PAMAM. Additionally, a sharp peak at 1205 cm−1 could be assigned to the NH group of amide groups in PAMAM, whereas it was weakened for HA because of steric hindrance of the HA chain. After HA was conjugated to the surface of PAMAM, N−H vibration signals were weakened in both HA-pep-PAMAM and HA−PAMAM, together with the strengthening of stretching of C−N (1046 cm−1) because of the introduction of HA in both HA-pep-PAMAM and HA−PAMAM in Figure S2. The 1H NMR spectra are shown in Figure S1, and the HA and AEM characteristic chemical shifts of the product, HA−AEM, appear in the spectrum, indicating that HA−AEM was synthesized, which is consistent with IR results. 3.2. Size-Shrinkable Characterization. After decorating PAMAM G5.0 with HA, the hydrated particle size of HA-pepPAMAM and HA−PAMAM increased to approximately ∼200 nm, which effectively increased the tumor accumulation because of the relatively large particle size.30 To study the shrinkable property of HA-pep-PAMAM, the particle size of HA-pep-PAMAM was measured after incubation with MMP-2 for 4 h at pH 7.4. As shown in Figures 3, S3, and S4, the no. 2 HA-pep-PAMAM in Figure S3A was selected for further studies because of its small particle size and good dispersity, and the transmission electron microscopy (TEM) images of HA-pepPAMAM/DOX showed distinguishing particle sizes and morphologies, which demonstrated significant differences in the particle size without/with MMP-2 digestion. The size of HA-pep-PAMAM/DOX showed a sharp transition from ∼200 to ∼10 nm in the presence of MMP-2, which indicates that HApep-PAMAM was size-shrinkable by MMP-2 digestion. However, no obvious changes were observed at the end of the incubation for HA−PAMAM, suggesting that these particles could not be degraded by MMP-2. Meanwhile, by increasing the incubation time, the cleavage of HA from HA-

samples were subjected to CLSM (OLYMPUS IX83-FV3000-OSR, Japan) with identical settings for each confocal study. 2.13. Statistical Analysis. All data are presented as mean ± SD. The statistical differences among the groups were analyzed using a one-way analysis of variance analysis. P < 0.05 was considered statistically significant.

3. RESULTS 3.1. Synthesis and Characterization of HA-pepPAMAM. The design and synthesis of DOX-loaded HA-pepPAMAM nanoparticles are shown in Figure 2, and their characterization is shown in Figure S1. The products were characterized by proton nuclear magnetic resonance (1H NMR) in D2O. Representative peaks belonging to PAMAMpep, HA−1-(2-Aminoethyl) maleimide (HA−AEM), HA− PAMAM, and HA-pep-PAMAM were observed in the 1H NMR spectrum of the nanoparticles in D2O, suggesting that the product was successfully synthesized. PAMAM-pep was synthesized by conjugating the amino group on PAMAM and the carboxyl group on the polypeptide. The 1H NMR spectrum is shown in Figure S1, and the characteristic chemical shifts of PAMAM and the polypeptide appear on the PAMAM-pep spectrum, indicating that the peptide has been introduced to the PAMAM structure. In addition, it is known that each PAMAM molecule contains 248 H in the NH2CH2CH2NHCOCH2− moieties, and their chemical shift is located at δ ≈ 2.3 ppm. There are 12 methyl protons on each polypeptide molecule with δ ≈ 0.8 ppm. The integration values of the two different proton types by MestReNova software were 1.00 and 0.31, indicating that ∼6−7 polypeptides were introduced to every PAMAM molecule, which is in accordance with the results of element analysis (data not shown). HA and AEM were subjected to reductive amination to produce the polymer HA−AEM. In the presence of sodium borohydride (NaBH4) buffer (pH 8.5), the six-membered ring at the end of the HA structure was condensed with the terminal amino group of AEM to form a Schiff base at high temperatures, which was then reduced by STAB to form a secondary amine. The IR spectrum shows characteristic 42463


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Figure 4. In vitro cell viability and uptake. (A) Cell viability of A549 after being incubated with DOX solution, HA−PAMAM/DOX, HA-pepPAMAM/DOX with or without Col pretreatment for 48 h. (B) Cell viability of MCF7 after being incubated with DOX solution, HA−PAMAM/ DOX, HA-pep-PAMAM/DOX with or without Col pretreatment for 48 h. (C) Cellular uptake of different formulations with A549 cells and MCF7 cells. Results are expressed as mean ± SD, n = 3.

(HA−PAMAM and HA-pep-PAMAM) for 4 h against these cells in monolayer culture, and the results are shown in Figures 4 and S5. The cytotoxicity of HA-pep-PAMAM/DOX (IC50, 0.299 μg/mL) against A549 cells was stronger than that of HA−PAMAM/DOX (IC50, 0.500 μg/mL). The cytotoxicities of HA-pep-PAMAM/DOX and MMP-2-pretreated HA-pepPAMAM/DOX were almost identical against A549 cells. This finding can be explained because A549 cells express MMP-2, so that an additional MMP-2 enzyme to degrade the peptide linker was not necessary. Regarding MCF-7 cells, the cytotoxicity of MMP-2-pretreated HA-pep-PAMAM/DOX (IC50, 0.760 μg/ mL) was significantly stronger than that of HA-pep-PAMAM/ DOX (IC50, 1.884 μg/mL). This is because MCF-7 cells express little MMP-2, so that an additional MMP-2 enzyme is required to degrade the peptide linker. 3.5. In Vitro Cellular Uptake. The MMP-2 highexpressing A549 and MMP-2 low-expressing MCF-7 cells were used to investigate the cellular uptake behavior of the DOX preparations by flow cytometry, and the results are shown in Figure 4. No significant difference was observed in the

pep-PAMAM increased gradually (Figure 3). These results suggest that the size reduction of HA-pep-PAMAM/DOX was caused by the cleavage of the MMP-2 substrate peptide. 3.3. Encapsulation and Release of DOX. The DOXloaded HA−PAMAM and HA-pep-PAMAM were prepared. HA−PAMAM and HA-pep-PAMAM were able to load 12.9 and 14.7% of DOX, respectively. The cumulative releases of DOX from HA−PAMAM/DOX and HA-pep-PAMAM/DOX were 92.2 and 85.1%, respectively, after incubating for 72 h (Figure S3B), suggesting that most of the DOX could be released from HA-pep-PAMAM/DOX or HA−PAMAM/DOX under physiological conditions. These results suggested that HA-pep-PAMAM/DOX possesses a controlled-release effect, enabling the sustained release of DOX in vivo. 3.4. In Vitro Cytotoxicity of DOX-Loaded Nanoparticles. We used MMP-2 poorly and highly expressed cancer cells (MCF-7 and A549, respectively) to assess the in vitro drug action. We examined the cytotoxicity of free DOX, HA−PAMAM/DOX, HA-pep-PAMAM/DOX, MMP-2-pretreated HA-pep-PAMAM/DOX, and blank nanoparticles 42464


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Figure 5. Penetration in A549 tumors. (A) Fluorescence distribution of A549 tumor spheroids after being incubated with the equivalent 4.0 μg/mL DOX of PAMAM/DOX(a), DOX solution (b), HA−PAMAM/DOX (c), HA-pep-PAMAM/DOX (d), and MMP-2 pre-treating HA-pep-PAMAM/ DOX (e) for 4 h, and the bar represents 200 μm. (B) Semi-quantitative intensity of the inner region at different sections of A549 tumor spheroids. (C) Frozen sections of tumors were harvested from A549 tumor-bearing mice at 24 h after administration with Cy5.5-labeled HA−PAMAM or HApep-PAMAM, then stained with an FITC-tagged CD31 antibody to label tumor vessels (green signal) or stained with DAPI to nuclei, followed by observation under CLSM (OLYMPUS IX83-FV3000-OSR, Japan).

uptake of HA-pep-PAMAM/DOX without enzyme pretreatment between A549 and MCF-7 cells, possibly because the incubation with drug solution was only for a short time and the A549 cells were unable to secrete sufficient enzyme to degrade the polypeptide linker. The uptake of MMP-2-pretreated HApep-PAMAM/DOX by the two cell types was significantly greater than that of HA-pep-PAMAM/DOX without MMP-2 pretreatment, which was because of the degradation of the peptide linker to release PAMAM/DOX and enhance uptake of the drug. 3.6. Tumor Spheroid Penetration. Cellular uptake may not accurately reflect nanoparticle tumor penetration because targeting delivery systems must be transported beyond the outer cell layers and into the cells in the deeper regions of solid tumors. The effect of different formulations in superficial sections of A549 tumor spheroids is shown in Figure 5A. The fluorescent intensity of HA−PAMAM/DOX and HA-pepPAMAM/DOX decreased dramatically inside of the spheroid, proving that these large particles failed to penetrate into the deeper part of the tumor spheroid. After being pretreated with MMP-2 for 4 h, HA-pep-PAMAM/DOX was largely discovered inside of spheroids, especially in the deep sections, indicating that smaller-sized particles had advantages in tumor penetration, which was consistent with other studies.8,31

To evaluate the MMP-2 sensitivity and the penetration of HA-pep-PAMAM/DOX, fluorescent intensities were measured in different parts of A549 tumor spheroids. The results are shown in Figure 5B, which are consistent with those mentioned above. By pretreating with MMP-2, the penetration effect of the HA-pep-PAMAM/DOX group into tumor spheroids was significantly enhanced, supporting the assumption that the shrinkage of particles was helpful during the penetrating process. We next harvested tumors from A549 tumor-bearing mice to evaluate the penetration efficiency of the nanocarriers and stained the tumor vessels with the endothelial marker CD31. As shown in Figure 5C, because a quantity of nanoparticles could accumulate in a tumor’s leaky blood vessels by the EPR effect, the insensitive nanoparticle HA−PAMAM could be observed, but most of them were still localized around the blood vessels, which indicates that a quantity of nanoparticles in the blood could passively extravasate through the leaky vessels caused by the EPR effect. Nevertheless, the enzyme-sensitive HA-pepPAMAM penetrated the tumor much farther away from the vessels because of the subsequently shrinkable size after its greater EPR effect. 3.7. Inhibition of Tumor Spheroids. The morphological characteristics of the tumor spheroids are shown in Figure S6A. 42465


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Figure 6. (A) In vivo optical real-time fluorescence imaging of nude mice bearing A549 tumors at 2, 6, 12, 24, and 48 h after intravenous administration of ICG, PAMAM/ICG, HA-pep-PAMAM/ICG, and HA−PAMAM/ICG at a dosage of 2.0 mg/kg of ICG. Fluorescence intensity was normalized to the same scale. (B) Representative ex vivo NIR fluorescence images of dissected organs of nude mice bearing A549 tumors at 48 h after intravenous administration of ICG, PAMAM/ICG, HA-pep-PAMAM/ICG, and HA−PAMAM/ICG. (C) Fluorescence signals of the excised organs at 48 h after intravenous administration.

Figure 7. Antitumor effects in nude mice bearing A549 tumors after being treated with PAMAM/DOX, HA−PAMAM/DOX, HA-pep-PAMAM/ DOX, DOX solution, and PBS. (A) Tumor growth curves after tail vein injection of PBS, DOX solution, PAMAM/DOX, HA-pep-PAMAM/DOX, or HA−PAMAM/DOX on nude mice bearing A549 tumors every 3 days for 6 times at a dosage of 2.0 mg/kg DOX (n = 6). (B) TUNEL staining of A549 tumors and hearts from mice treated with different groups. (C) Image of A549 tumors extracted from the mice at the end of the experiment. (D) Average weight of A549 tumors treated with different groups, *p < 0.05, **p < 0.01.

Figure 6. Regarding free ICG and the ICG-loaded PAMAM group, strong fluorescence was observed in the hypogastrium, and both ICG signals were quickly cleared by 24 h. By contrast, a systemic distribution was found in the ICG-loaded HA-pepPAMAM and HA−PAMAM nanoparticle-treated groups. Over time, stronger fluorescence signals were observed in the tumor sites, which indicated that more ICG-loaded nanoparticles were accumulating in the tumors. At 48 h, the organs and tumors were harvested (Figure 6B), and the images revealed that the lung had the strongest fluorescence after injection with free ICG. For the ICG-loaded nanoparticles, the tumor showed the strongest fluorescence (Figure 6C). These results were in accordance with the real-time distribution. The distribution of ICG changed significantly after encapsulation by HA−PAMAM or HA-pep-PAMAM. The tumor-targeting property of the HApep-PAMAM or HA−PAMAM nanoparticles would be favorable for in vivo therapy. 3.9. Antitumor Effect. Cultured in various formulation for 48 h, the viability of A549 cells decreased with the increasing

The inhibition results of A549 tumor spheroids are shown in Figure S6B. In the presence of DOX solution or the DOXloaded HA-pep-PAMAM nanoparticles, the structure of the tumor spheroids was not compact, with cells dislodging from the edges and appearing apoptotic. Compared with HA− PAMAM/DOX, HA-pep-PAMAM/DOX had an inhibitory effect on the growth of tumor spheroids. The volume of the tumor spheroids treated with free DOX was 53.1 ± 12.1% after 6 days. Meanwhile, the group treated with the DOX-loaded HA-pep-PAMAM had a slightly smaller volume (73.1 ± 6.4%) than the DOX-loaded HA−PAMAM group (85.7 ± 11.2%) did on day 6. All groups showed lower volumes than that of the control group (135.9 ± 18.9%). 3.8. In Vivo Distribution. ICG is one of the tricarbocyanine dyes, which is widely used in bioimaging in vivo because of its deep tissue penetration, minimum background interference, and low accumulation in tumors in its free form.32,33 The in vivo distribution of free ICG, PAMAM/ICG, and ICG-loaded nanoparticles is shown in 42466


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Figure 8. Biochemical parameters of ALT (A), AST (B), LDH (C), and CKMB (D) in the serum of mice administrated with DOX preparations for 21 days. Results were presented as mean ± SD (n = 4). *p < 0.05 vs the control, **p < 0.01 vs the control, and ***p < 0.001 vs the control. PBS was used as control.

the treated animals. The results are shown in Figure 8C,D. Elevated levels of LDH were found in the free DOX and the PAMAM/DOX group, showing their cardiotoxicity, whereas the LDH levels of the DOX-loaded HA−PAMAM or HA-pepPAMAM groups were not significantly different compared to those of the PBS or HA-pep-PAMAM groups. 3.11. Immunohistochemistry Analysis. Histological analysis of H & E-stained organs and tumors was performed, and the results are shown in Figures S8−S13. Cardiotoxicity is one of the serious adverse effects of DOX,34 which limits its clinical application. The histopathological differences are illustrated in Figure S8. Cardiomyocytes in the DOX solution group are slightly sparse and unevenly arranged, indicating that there was myocardial cell damage. The PAMAM/DOX group also shows obvious cardiotoxicity because the myocardial cells are disordered, and the size of the nuclei varied. Compared with that of the PBS and PAMAM/DOX groups, there was no obvious histopathological abnormality in the myocardium of the HA−PAMAM/DOX or HA-pep-PAMAM/DOX groups, which indicates that DOX encapsulation by the HA-modified nanoparticles can reduce the cardiotoxicity caused by free DOX. Liver is the main organ for metabolism in the body. Because of their particle-like properties, nanoparticles can easily accumulate in the liver. When a large amount of drug accumulates in the liver, it is likely to cause hepatotoxicity. The normal liver tissue sections in Figure S9 show regular cell arrangements and visible nuclei, while the cells are necrotic, with inflammatory cell infiltration, in the DOX solution group. In the PAMAM/DOX group, the cells vary in size, accompanied by nuclear pyknosis, cell rupture, and inflammatory cell infiltration. By contrast, the HA−PAMAM/DOX and HA-pep-PAMAM/DOX groups have normal liver cells and the nuclei are clear, indicating that both nanopreparation groups are not toxic to the liver, which is consistent with the results of the blood biochemical analysis. Through H & E staining, HA-pep-PAMAM/DOX-treated A549 tumors demonstrated obvious pathological changes, undergoing more necrosis and apoptosis compared to other groups (Figure S13). Meanwhile, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining of A549

DOX concentration (Figure 4A,B), inducing the apoptosis of A549 cells in vitro. Nevertheless, the antitumor effect on tumor-bearing mice varied significantly after several injections of these formulations. By measuring the tumor volume, the antitumor effect of different formulations was measured (Figure 7A). The tumor inhibition rate (62.5%) of the HA-pepPAMAM/DOX group was much higher than that of the HA− PAMAM/DOX group (26.1%, P < 0.01), free DOX group (50.2%, P < 0.05), and PAMAM/DOX group (28.9%, P < 0.01). After the last treatment, the HA-pep-PAMAM/DOXtreated group was much smaller in average tumor volume and weight, demonstrating that HA-pep-PAMAM/DOX exhibited an excellent antitumor efficiency, which was further highlighted by the tumor images (Figure 7C). Additionally, the body weights of the mice in each group were determined, and they are summarized in Figure S7. The body weights decreased gradually during the injection with free DOX (7.6%) or PAMAM/DOX (12.4%), which was caused by the serious side effects of DOX and PAMAM.34,35 All these in vivo antitumor experiments demonstrate that this strategy of enzyme-triggered penetration was applicable in deep tumor tissues and can be further testified for other tumor treatments. 3.10. Blood Biochemical Analysis. ALT and AST mainly exist in the nucleus and mitochondria. When liver cells are significantly damaged, these enzymes are released into the blood. Therefore, increased levels of ALT and AST in the blood are specific indicators of liver damage and are used to diagnose liver damage.36,37 Figure 8A,B shows the mean serum ALT and AST values from the different treatment groups. Regarding the free DOX and PAMAM/DOX groups, ALT and AST levels were significantly higher than those in the PBS group, indicating abnormal liver function in both treatment groups. By contrast, the levels of ALT and AST in the HA-pepPAMAM/DOX- and HA−PAMAM/DOX-treated groups were not different from those of the PBS or HA-pep-PAMAM group, demonstrating that both DOX preparations do not cause hepatotoxicity. The elevation of serum levels of enzymes due to myocardial necrosis, such as LDH and CK-MB, is considered a marker of myocardial infarction. This study was also designed to determine the changes in the levels of LDH and CK-MB in 42467


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vitro,49,50 these penetrating agents have not been reported to elicit clinical efficacy because of their nonspecific nature. The particle size is vital in determining the penetration ability of nanoparticles into the tumor site, as diffusion is inversely proportional to the particle size. It has been demonstrated that larger nanoparticles, despite being more suitable for improved pharmacokinetics and vascular extravasation, are inherently unfavorable to tumor penetration because of their massive diffusion barrier in the dense tumor matrix. This explained why the clinically approved DOXIL (∼100 nm size) showed only little competence in therapeutic efficacy. On the contrary, smaller nanoparticles excelled in tumor penetration but generally had short half-lives and insufficient tumor accumulation because of their rapid clearance. Therefore, an ideal drug delivery system should have a relatively large size initially to obtain selective accumulation and longer circulation, and it should be degraded to smaller particles at tumor sites to enhance penetration. Such dilemmas have contributed to the development of stimuli-responsive nanoparticles that their size are shrinkable in response to pH, enzymes, or UV light. However, the means that were employed to trigger size alterations can be problematic in the clinic. For example, UV light has poor tissue penetration and is harmful to normal tissues,51 which seems to be impractical in clinical practice. For the pH-triggered size-switchable systems, their size alterations involve a sharp conversion between hydrophilicity and hydrophobicity and usually take hours to complete,52 which may reduce the penetrating capability of small particles and compromise treatment efficacy. Compared to normal tissues, many enzymes are overexpressed in the pathological state, such as proteases, glycosidases, MMPs, lipases, and phospholipases. MMPs are zinc-dependent endopeptidases that can degrade many ECM proteins. Studies have shown that many MMPs are overexpressed and active during the advanced cancer, whereas they are minimally expressed in normal tissue. This ingenious design ensured drug release only occurred when it is needed. In particular, MMP-2 and MMP-9 (type IV collagenases), which are able to degrade the ECM, are important substances involved in tumor progression, angiogenesis, and metastasis. In recent years, a series of smart nanoplatforms have been developed via the introduction of several specific enzyme substrates to the structure of nanoscale formulations.52 Combined with the superiority of stimuli-responsiveness and nanotechnology, enzyme-responsive systems allow precisely controlled drug delivery and release. In such designs, nanoparticles can aggregate in the targeted tissue or reach specialized cellular organelles, where the drug is locally released after the cleavage of labile linkages by enzymes. In the present study, the MMP-2-sensitive peptide, PLGLAG,53 was introduced to afford the nanoparticles the ability to shrink their size after the target site has been reached to release DOX physically entrapped in PAMAM and improve the penetration and retention in tumor tissue. The difference between our nanoparticles and other MMP-2-sensitive carriers that were reported previously is that PAMAM, which has a small particle size, can also be used as a drug carrier because of its spherical, hydrophobic structure. In addition, the positive charges on its surface are beneficial for penetrating the tissues, and it usually can be combined with a gene of interest to form a multifunctional drug delivery platform. Combined with HA, which is a natural polysaccharide and plays an important role during tumorigenesis, the particles are given a good

tumors was used to evaluate the degree of apoptosis, and the results are shown in Figure 7B. More apoptotic bodies was found in tumors for mice receiving HA-pep-PAMAM/DOX treatment than that for mice receiving other treatments, indicating that injection with HA-pep-PAMAM/DOX leads to more tumor cell apoptosis. Meanwhile, compared with that of the PBS group, the DOX solution group produced slightly more myocardial apoptosis. These results indicate that HA-pepPAMAM/DOX preparations can also reduce DOX cardiotoxicity, while achieving antitumor efficacy.

4. DISCUSSION Low penetration of anticancer drugs into many tumors remains an important factor, limiting their therapeutic efficacy. A variety of factors, including the delivery by the vascular system and the rate of diffusion through the tissue, can influence the distribution of smaller-molecular-weight drugs and larger nanoparticle systems in tumors. Antineoplastic drugs usually get access to solid tumors through the blood supply and must penetrate the extravascular space to reach all the cancer cells in a concentration sufficient to kill the cells. The high variable leakiness of the vascular endothelium in solid tumors38 and the increased interstitial pressure39,40 with the increasing distance from the vessels further result in a poor penetration effect of nanoparticles to the tumor deeply. In addition, the dense ECM is deemed an additional obstacle, with solid tumors being more difficult to penetrate. All these factors cause many anticancer drugs to penetrate only 3−5 cell diameters from the blood vessels.41 Additionally, fibrillar collagens such as types I and III42,43 in the interstitial matrix increasingly accumulated in other tumors, which may limit nanocarriers, especially the large one, diffuse through the interstitial matrix. Extensive efforts have been made to overcome the penetration obstacles inherent in nanoparticle systems. Traditionally, it was proved that a limited tumor blood supply could inhibit tumor growth. Such therapeutic strategies, therefore, have been applied clinically for years, but their effects are disputable on account of drug resistance. 44 Excessive production of the angiogenesis factor led to abnormal and dysfunctional tumor vessels. According to these studies, Jain have proposed a method that “normalizes” the abnormal blood vessels in tumors by reducing their leakage; this has been shown to reverse the abnormal state of blood vessels, to improve the delivery of oxygen and nutrition, and to reduce IFP.45,46 Although they have enhanced the effect of existing antiangiogenic therapies (e.g., sorafenib, a tyrosine kinase receptor inhibitor),46,47 the application is limited by the need to counteract the potential negative impact on the EPR effect. There is also a risk of accelerating tumor progression and even metastasis by uncontrolled degradation of the matrix. One of the major culprits for high IFP is the ECM, which mainly consists of collagen, elastin fibers, proteoglycans, and glycosaminoglycans and creates a gel-like medium that is difficult to traverse with the result that the nanoparticles failed to get across 70−100 nm or larger space between ECM fibers. The ECM structure, and therefore the IFP, can be changed by matrix-degrading enzymes. For example, co-expression of hyaluronidase enhances the antitumor activity of Ad in vivo and induced tumor regression as a result of increased distribution of Ad in the tumor site.48 Additionally, although cell penetrating peptides or protein transduction domains are highly efficient in mediating the cellular uptake of drugs in 42468



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biocompatibility and a larger particle size to achieve longer blood circulation half-lives. Although the overall nanocarrier structure is simple, it can be used as a promising platform, which can be used for tumor diagnostic imaging and targeting treatments when combined with drugs, genes, or other imagining molecules.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b10098. 1 H NMR spectrum of synthesis of HA-pep-PAMAM in D2O; FTIR spectra of HA, PAMAM, HA−PAMAM and HA-pep-PAMAM; characterization of HA-pep-PAMAM; accumulated release of DOX in PBS at 37 °C; TEM image of HA−PAMAM; in vitro cytotoxicity of HA− PAMAM and HA-pep-PAMAM in A549 and MCF7; inhibition of DOX on tumor spheroids; the body weight changes after tail vein injection of PBS, DOX solution, PAMAM/DOX, HA-pep-PAMAM/DOX, or HA− PAMAM/DOX in nude mice; histological alterations in hearts, livers, spleens, lungs, kidneys, and tumors at 21 days after tail vein injection of different DOX preparations (H & E staining, ×20) (PDF)

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REFERENCES

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5. CONCLUSION Herein, a novel tumor microenvironment-responsive drug delivery system was established. With relatively longer blood circulation, the obtained HA-pep-PAMAM/DOX was relatively large in size and better in selected extravasation via the EPR effect. Crossing the leaky tumor vessel, HA-pep-PAMAM/ DOX penetrated into tumor tissues and degraded by the highly overexpressed MMP-2. The resulting PAMAM/DOX with a small size possessed a better interstitial penetrating efficiency, mostly accumulated into a deeper tumor region and absorbed by tumor cells in the tumor microenvironment. In combination, HA-pep-PAMAM/DOX presented an excellent tumor-penetrating efficiency and significant antitumor activity. Therefore, the enzyme-sensitive strategy will open a new era in the further exploration of delivery systems for enhancing drug penetration and retention in the tumor site.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Min Han: 0000-0001-9373-8466 Jian-Qing Gao: 0000-0003-1052-7060 Author Contributions §

M.H. and M.-Y.H.-F. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by National Basic Research Program of China (no. 2014CB931901), National Natural Science Foundation of China (81373346, 81572952, 81673022), and Fundamental Research Funds for the Central Universities (2017XZZX011-04). 42469


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