MMP-2-Sensitive HA End-Conjugated Poly(amidoamine) Dendrimers

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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, Ningning Guo, Jiejian Chen, Huina Liu, Zhiqi Xie, Mengting Lin, Qichun Wei, and Jianqing Gao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b10098 • Publication Date (Web): 16 Nov 2017 Downloaded from http://pubs.acs.org on November 16, 2017

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MMP-2 Sensitive HA end-conjugated poly(amidoamine) dendrimers via click reaction to enhance drug penetration into solid tumor Min Hana,#, Ming-Yi Huang-Fua,#, Wang-Wei Guoa, Ning-Ning Guoa, JieJian Chenb, Hui-Na Liua, Zhi-Qi Xiea, Meng-Ting Lina, Qi-Chun Weib, Jian-Qing Gaoa,* a

College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, P.R.China.

b

Department of Radiation Oncology, Key Laboratory of Cancer Prevention and Intervention, the Second Affiliated Hospital, Zhejiang University,

College of Medicine, Hangzhou, 310058 China.

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) due to cleavage of PLGLAG. This rapid size-shrinking characteristic not only promoted nanoparticle extravasation and accumulation in tumors benefited from the enhanced permeability and retention (EPR) effect, but also achieved faster nanoparticle diffusion and penetration. We have further conducted comparative studies of MMP-2-sensitive macromolecules (HA-pep-PAMAM) and MMP-2-insensitive macromolecules (HA-PAMAM) synthesized with similar particle size, surface charge, and chemical composition, and evaluated in both monolayer cells and multicellular spheroids. The results confirmed that the enzymeresponsive 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 also can 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 Cancer is one of the most serious health threats for humans, which has aroused great concern.1 Currently chemotherapy is considered effective approach for treating human cancers,2 but the existence of systemic toxicity, lack of tumor targeting, and tumor multidrug resistance limit 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 minimizing systemic toxicity. To obtain effective tumor therapy, drugs must penetrate deeply tumor tissue 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 therapeutic drugs are significantly blocked from entering adjacent areas of the tumor vasculature after extravasation from the tumor

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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 In order to address the dilemma of improving the efficacy of treatment, 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 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 PAMAM dendrimers, we firstly designed a size-shrinkable drug delivery system responsive to the tumor microenvironment based on a polysaccharide-modified dendrimer in present study,13-14 which contains poly(amidoamine) (PAMAM), hyaluronic acid (HA), and a matrix metalloproteinase-2 (MMP-2)-sensitive peptide linker (PLGLAG), using doxorubicin (DOX) as a model drug. PAMAM is one of the most widely studied dendrimers.15-16 However, due to 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 due to 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 -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, over modification of the carboxylic acid groups in 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. The terminal end of the 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. 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 the HA-pep-PAMAM/DOX, using A549 and MCF-7 cells. A schematic diagram (Fig. 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. 2. EXPERIMENTAL SECTION 2.1. Materials and Animals. Doxorubicin Hydrochloride (DOX) was purchased from Zhejiang Hisun Pharmaceutical Co. Ltd. (Zhejiang, China), and hyaluronic acid (HA, MW=17KDa, 48KDa) was provided by

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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). 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 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-EDTA solution (0.5% trypsin, 5.3Mm EDTA tetrasodium) was purchased from Jinuo Biomedical Technology Co., Ltd(Hangzhou, China), and fetal bovine serum was provided by Thermo Fisher Scientific (Waltham, MA, USA). CCK8-assay kit were purchased from Beyotime Biotechnology Company (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 Co.(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 (the 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 standard housing conditions and all related experiments were conducted according to guidelines which has 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, 0.3mol/L sodium chloride). Then 30.5 mg ethylmethyl maleimide (AEM) and 25.4 mg sodium triethoxyborohydride (STAB) were added to the HA solution and stirred at 60 °C for 4 days in water bath. Subsequently, the product solution was dialyzed against water to remove unreacted substance. The purified HA-AEM was lyophilized and stored at -20 °C. PAMAM G5.0 (28.0mg) was dissolved in distilled water (2.0mg/mL). Peptide (6.9mg) was dissolved in double distilled water. After addition of activator and 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 for 4 h. The product solution was dialyzed against water to remove unreacted substance. The purified PAMAM-pep was lyophilized and stored at -20 °C. The products above were mixed in 1:1 (mass ratio) and stirred for 4 h at 0.2 M PBS (pH 7.4) at room temperature, then was dialyzed for 48 h against PBS to give HA-pep-PAMAM. 2.3. Synthesis of HA-PAMAM. PAMAM G5.0 (28.8mg) 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 hyaluronic acid was completely dissolved. Then NaBH3CN (25.4mg) was added to the reaction solution and then stirred at 60 ° C for 4 days in water bath. Subsequently, the product solution was dialyzed against PBS to remove unreacted substance. The purified HA-PAMAM was stored at 4 ° C. 2.4. Cleavage of HA-pep-PAMAM by MMP-2. HA-pep-PAMAM nanoparticles (4mg/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 CTAB method.18-19 In addition, to

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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. Nanoparticles Characterization. The morphology of HA-pep-PAMAM/DOX (with/without MMP-2 pretreatment), HA-PAMAM/DOX were captured by transmission electron microscope (TEM, JEM-1230, JEOL, Japan). The zeta potential and particle size were analyzed by dynamic light scanning (DLS) analysis using a zetasizer nano ZS (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 doxorubicin 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 0h to 96 h. All procedures were carried out in the dark. 2.6. Cellular Uptake Study. For cellular uptake study, the cells were seeded in six-well plates at a cell density 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 4h 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 0.5mL PBS, followed by intensity analysis on a flow cytometer (AccuriTM C6 flow cytometer ,BD Biosciences). The data were collected and analyzed using AccuriTM 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) 20and MCF-7 cells without MMP-2 overexpression21-22 were grown at about 3 × 103 cells/well in 96-well plates. Cells were incubated with DOX·HCl solution, PAMAM/DOX, HA-PAMAM/DOX, HA-pep-PAMAM/DOX, or MMP-2-treated HA-pep-PAMAM/DOX at various drug concentrations (0.0625-2µg/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 the 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 100rpm 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 (200µL) 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)

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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-pep-PAMAM/DOX, HA-PAMAM/DOX, or HA-pep-PAMAM/DOX pretreated with MMP-2 for 4h. After culturing for 4 h, the tumor spheroids obtained were washed with cold PBS for 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). Tumor spheroids size was measured and morphology was recorded every day during the study. The tumor spheroids 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 post injection, the biodistribution of the fluorescent particles was observed in vivo. At 48h, the mice were sacrificed, and the tumor and organs (heart, liver, spleen, lung, and kidney) were harvested and then imaged using the MaestroTM in vivo imaging system (Cambridge Research and Instrumentation, USA). 2.11. Anti-tumor Effect. A549 tumor bearing mice were established as described above. One weeks after implantation, the mice were randomly divided into 6 groups (6 mice per group): PBS, DOX solution, PAMAM/DOX, HA-pep-PAMAM/DOX, HA-PAMAM/DOX and HA-pep-PAMAM. Each A549 tumor bearing 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 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 creatinekinase-MB (CKMB) 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 heart, liver spleen, lung, kidney, and tumor was 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 histological study of toxicity. Additionally, sections of 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 intravenous 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%

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paraformaldehyde for 10 min and blocked with 10% bovine serum albumin (BSA) for 1 h, followed by incubation with rat anti-CD31 antibody (1:50) overnight at 4°C. Tumor slices were washed 3 times with PBS and incubated with a fluorescein isothiocyanate (FITC)-conjugated goat anti-rat immunoglobulin G (IgG) secondary antibody (1:100) and 4',6-diamidino-2-phenylindole (DAPI) for 3 h, then rinsed and sealed with a coverslip. The samples were subjected to confocal laser scanning microscopy (OLYMPUS IX83-FV3000-OSR, Japan) with identical settings for each confocal study. 2.13. Statistical Analysis. All data were presented as mean ± standard deviation (SD). The statistical differences among the groups were analyzed using a one-way ANOVA analysis. P