PEGylated Cationic Vectors Containing a Protease-Sensitive Peptide

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PEGylated Cationic Vectors Containing a Protease-sensitive Peptide as a miRNA Delivery System for Treating Breast Cancer Ye Zeng, Zixuan Zhou, Minmin Fan, Tao Gong, Zhirong Zhang, and Xun Sun Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00726 • Publication Date (Web): 27 Nov 2016 Downloaded from http://pubs.acs.org on November 29, 2016

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Molecular Pharmaceutics

PEGylated Cationic Vectors Containing a Protease-sensitive Peptide as a miRNA Delivery System for Treating Breast Cancer Ye Zeng, Zixuan Zhou, Minmin Fan, Tao Gong, Zhirong Zhang, Xun Sun* Key Laboratory of Drug Targeting and Drug Delivery Systems, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu, Sichuan, P.R.China. *Correspondence: Xun Sun, West China School of Pharmacy, Sichuan University No. 17, Section 3, Southern Renmin Road, Chengdu 610041, People’s Republic of China Tel/Fax: +86-28-85502307 E-mail: [email protected]

KEYWORDS: MMP2-cleavable, Gene vector, MiR-34a, Breast cancer

GRAPHICAL ABSTRACT

ABSTRACT Several targeted drug delivery systems have recently been developed to increase the bioavailability of a drug at its site of action, allowing simultaneous reduction of the total necessary drug dose as well as side effects. Here we designed a cationic gene 1

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vector containing matrix metalloproteinase-2 (MMP2)-cleavable substrate peptides that specifically targets tumor sites, where MMP2 levels are high. The targeted delivery system is fabricated by linking enzyme-cleavable polyethylene glycol (PEG) derivatives to cationic β-cyclodextrin-polyethyleneimine conjugates, which reduce the toxicity of polyethyleneimine and condense the therapeutic cargo. In the present study, tumor suppressor microRNA miR-34a, which suppresses onset and progression of many types of cancers, was investigated for its therapeutic potential for treating breast cancer. The PEG coating markedly reduces nonspecific interaction between cationic particles and serum proteins, permitting accumulation at the target site; subsequent peptide cleavage by MMP2 facilitates miR-34a delivery into tumor cells. The nanopreparation showed excellent stability, and its internalization, tumor targeting and anti-tumor efficacy in vitro and in vivo were better than those of a nanopreparation containing MMP2-uncleavable peptide.

1. INTRODUCTION Researchers have experimented with numerous approaches to improve the selectivity of tumor treatment by developing triggers based on physical stimuli, pH, ionic strength, redox potential, temperature, up-regulated proteins, and magnetic fields;1-4 many of these triggers reflect known characteristics of the tumor microenvironment. Perhaps the most commonly used approach is to modify the surface of drug nanocarriers with targeting moieties such as monoclonal antibodies, peptides, and small molecules. Peptide conjugation has proven particularly powerful for developing useful biomaterials.5-7 Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases overexpressed in the microenvironment of tumor regions, and MMP-mediated degradation of the extracellular matrix leads to cancer cell invasion and metastasis.8 Breast cancer, one of the most malignant tumor types, acquires the ability to invade the underlying basal lamina and its adjacent stroma, allowing it to metastasize to other tissues.9 High MMP expression is associated with development of breast cancer.9-11 Numerous substrates capable of being recognized and cleaved by MMPs, particularly MMP2, have been developed for targeted drug delivery and tumor imaging.3, 4, 12-14 Nevertheless, delivery of MMP2-cleavable drugs has rarely been reported in the treatment of breast cancer. Gene therapy holds promise for treating a wide range of diseases, and in the case of treating cancer, four main strategies have been used: turning off oncogene expression, enhancing tumor suppressor expression to induce apoptosis of cancer cells, inhibiting neoangiogenesis, and stimulating the anti-tumor immune response.14 Oncogenes and tumor suppressor genes play a crucial role in cancer onset and progression,15 and targeting these genes may allow the treatment of tumors where they originate. A class of molecules recently implicated in cancer is micro-RNAs (miRNAs), which are endogenous, non-coding, single-stranded RNAs of 19-25 nucleotides. They are transcribed as pre-mRNAs, cleaved by endonuclease and transported to the 2


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nucleus, where they mature into miRNAs that regulate the expression of genes involved in cell differentiation, proliferation, apoptosis, and the stress response.16 The miRNA miR-34a is an important mediator of p53 activity, and it is transcribed in the 1p36 region, which frequently suffers hemizygous deletion in human neuroblastomas and various other cancers.17 Levels of miR-34a are lower in triple-negative and mesenchymal breast cancer cell lines than in healthy breast tissue,18 and miR-34a may inhibit prostate and melanoma cancer stem cells by targeting CD44.19, 20 These findings raise the possibility that administering a synthetic mimic of miR-34a to tumor cells to restore expression of this miRNA might induce cell apoptosis and block tumor proliferation.21 Gene vectors constructed from cationic materials such as cationic polymers, lipids, dendrimers, and proteins, have shown great promise in delivering gene medicines effectively and achieving target-specific interference in vitro and in vivo.22 It may be easy to condense miRNAs into nanoparticles via electrostatic interactions, and these particles are known to efficiently enter target cells by endocytosis, membrane fusion, or both.2 On the other hand, cationic vectors can bind to negatively charged serum proteins and then promote aggregation and perhaps trigger phagocytosis, preventing accumulation at the target sites or stimulating production of inflammatory cytokines.23 This nonspecific protein adsorption can be reduced by incorporating hydrophilic moieties such as PEGs into gene vectors. In this study, we designed an MMP2-cleavable PEGylated gene vector and examined whether it was taken up by cells better than the corresponding non-PEGylated cationic vector or MMP2-uncleavable vector, and whether it accumulated to a greater extent at tumor sites where high levels MMP2 were present. The MMP2-cleavable PEGylated gene vector was prepared by synthesizing the hydrophilic copolymer CD-PEI2K, in which β-cyclodextrin serves to reduce the cytotoxicity through lower the charge density of polyamine backbone as well as increase the biocompatibility of cationic PEI vectors. We modified PEG with the MMP2 substrate peptide GPLGIAGQ, and conjugated this to the carrier. The resulting cationic vector CD-PEI-GPLGIAGQ-PEG2000 was complexed with miR-34a to yield the nanopreparation En-CNP, which should be cleaved by the abundant MMP2 at tumor sites. This cleavage should expose additional positive charges and thereby enhance nanoparticle internalization and anti-tumor efficacy. We validated the selectivity and efficacy of this delivery system in mice bearing xenografts of 4T1 triple-negative breast cancer tumors that express high levels of MMP2.

2. EXPERIMENTAL SECTION 2.1. Materials and Cells. Polyethylenimine (PEI, branched, MW 2 kDa), β-cyclodextrin (β-CD, MW 1134), N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), 1,1’-carbonyldiimidazole (CDI), gelatin, polyacrylamide, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Cleavable PEG2K-MMP2 peptide 3



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(GPLGIAGQC) and uncleavable PEG2K-MMP2 peptide (IPGQGALGC) were synthesized by Leon Chemical (Shanghai, China). Human MMP2 was purchased from Sino Biological (Beijing, China). The miRNA-34a mimic 5’-UGGCAGUGUCUUAGCUGGUUGU-3’ and the corresponding FITC- or CY5-labeled versions were synthesized by RUIBO (Guangzhou, China). All other reagents were of analytical purity and were used as received. 4T1, HT1080, MDCK cells obtained from ATCC Shanghai Cell Institute, Chinese Academy of Sciences. 4T1 and HT-1080 cells were cultured at 37 °C in DMEM containing 10% FBS and 1% penicillin/streptomycin in incubation with a 5% CO2 atmosphere. A549 cells were cultured in complete RPMI 1640 medium. 2.2. Gelatin Assay Gelatin zymography was used to screen for a cell line that expressed sufficient MMP2 to allow us to test the selectivity of our nanodelivery system. 4T1, HT1080 and MDCK cells were incubated with serum-free medium for 48 h, and the conditioned medium was collected, cell debris was removed and MMPs were concentrated using a centrifugal filter device (Amicon® Ultra-15 with 30 kDa molecular weight cut-off, Millipore, USA). Concentrated proteins were separated on an SDS-PAGE gel containing 1 mg/mL gelatin (60 mV in stacking gel, 100 mV in resolving gel). Purified commercial human MMP2 served as a migration standard. After electrophoresis, the gel was incubated in 2.5% Triton X-100 for 60 min, followed by 30 min in buffer containing 50 mM Tris-HCl (pH 7.5) and 5 mM CaCl2, and finally 18 h in buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM CaCl2, and 1 mM ZnCl2. Aftewards the gel was stained with 0.25% Coomassie Brilliant Blue R-250 and destained in 12.5% methanol/10% acetic acid. Gel images were obtained using the ChemiDocTM 219 XRS system (Bio-Rad, USA). 2.3. Synthesis of CD-PEI2K, CP-C-PEG, and CP-unC-PEG CD-PEI was synthesized as follows: β-cyclodextrin (1.68 g, 1.48 mmol) was dissolved in N, N-dimethylformamide (DMF) (24 mL). Then CDI (3.21 g, 19.8 mmol) was quickly added to the DMF and stirred at 25 °C for 2 h under nitrogen. The resulting CD-CDI was precipitated in cold diethyl ether for purification. The purified product was then dissolved in 20 mL dimethylsulfoxide (DMSO) and stored at 4 °C until the next step. PEI2K (0.784 g, 0.44 mmol) was dissolved in DMSO (16 mL) and triethylamine (Et3N) (0.3 mL) was added to this PEI2K solution. Afterwards, 1/8 of CD-CDI obtained above was added dropwise to the PEI2K solution with stirring. After 12-h stirring, the solution was dialyzed against distilled water for 3 days and lyophilized overnight. The prepared CD-PEI (28.6 mg) was dissolved in PBS-EDTA solution (4 mL) containing 100 mM sodium phosphate (pH 7.5), 150 mM NaCl, 1 mM EDTA and 0.02% sodium azide. Then DMSO (200 µL) containing SPDP (2.30 mg, 7.4 µmol) was added. The mixture was stirred for 30 min at 25 °C, then divided into two halves. To one half was added PEG2000-MMP2-cleavable peptide (33.18 mg) and to another half was added PEG2000-MMP2-uncleavable peptide (33.23 mg); in each case, 4


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peptide was dissolved in PBS-EDTA (3 mL) and added dropwise to the bulk solution. The resulting mixtures were stirred overnight at 25 °C, then dialyzed thoroughly against distilled water for 3 days and lyophilized. The structures of CD-PEI2K, CP-C-PEG and CP-unC-PEG were characterized by 1H NMR (600 MHz; Bruker Corporation, Germany). 2.4. Preparation of CP2K, En-CNP and En-unCNP Nanoparticles Nanoparticles were prepared by adding miR-34a solution (1 mg/mL) to gene vector solution (5 mg/mL) at various PEI/miRNA ratios (w/w), followed immediately by vortexing. The resulting nanoparticles were allowed to stand at room temperature for 30 min before use. Nanoparticles used in subsequent experiments were prepared by mixing CP2K, En-CNP or En-unCNP vectors with miR-34a at a ratio of 4:1 (w/w) to give, respectively, CP2K (CD-PEI2K/miRNA), En-CNP (CP-C-PEG/miRNA) and En-unCNP (CP-unC-PEG/miRNA). Size distribution and zeta potential were measured by dynamic light scattering using a Zetasizer Nano ZS90 (Malvern Instruments, UK). Transmission electron microscopy was used to study the morphology of nanoparticles. Particles were dispersed onto a copper grid, stained with phosphotungstic acid (1%) for 20 s, then observed using an H-600 transmission electron microscope (Hitachi, Japan). 2.5. Interaction of Nanoparticles with Serum Different nanoparticles carrying CY5-miRNA were prepared and aliquots (20 µL) were mixed with murine serum (180 µL; 1:9, v/v) and incubated for 2 h, then centrifuged at 2200 rpm for 10 min. The supernatant was collected (50 µL) and mixed with an equal volume of phosphate-buffered saline (PBS). CY5 fluorescence in the mixture was determined by measuring the emission at 670 nm under excitation at 644 nm using a Varioskan Flash microplate reader (Thermo-Scientific, USA). CY5-miR-34a alone was used as a blank control. Each sample was measured in triplicate. 2.6. MTT Assay 4T1 cells were seeded in 96-well culture plates and allowed to reach approximately 80% confluence, at which point we treated them with different concentrations of PEI25K, CD-PEI2K, CP-C-PEG or CP-unC-PEG for 4 h with serum-free medium. Then the medium was replaced and cells were incubated for 24 h in complete medium. Cytotoxicity of the three materials was assessed using the MTT method (n = 3). MTT (10 µL, final concentration of 0.5 µg/mL) was added to each well and incubated for 4 h at 37 °C. Then we carefully removed the supernatant, incubated the wells at 37 °C for 30 min in dimethyl sulfoxide (DMSO, 150 µL) to dissolve the formazan crystals, and measured absorbance at 570 nm. 2.7. Uptake of Nanoparticles by Cells In Vitro 4T1 cells were seeded in 12-well dishes at 1.0 × 105 cells/well, and their internalization of nanoparticles was quantified using flow cytometry. When cells 5



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reached 80% confluence, half the culture medium was removed, and 500 µL of medium containing different types of nanoparticles loaded with FITC-miRNA was added (equivalent to 2 µg miR-34a), and cells were incubated for 2 h at 37 °C. Then cells were harvested, washed three times in cold PBS and digested with trypsin to yield a suspension. The percentage and the fluorescence intensity of cells was determined using a flow cytometer (Beckman Coulter, USA) equipped with a 488-nm argon laser for excitation. Uptake of nanoparticles was also examined within individual cells using confocal laser scanning microscopy. Cells were seeded onto glass coverslips in 6-well dishes (1 × 105 cells per coverslip) and incubated overnight. Half the culture medium was removed and cells were cultured for 2 or 4 h with CP2K, En-CNP, or En-unCNP loaded with CY5-miRNA (500 µL, equivalent to 2 µg miR-34a), washed three times with cold PBS, fixed with 4% (w/v) paraformaldehyde for 10 min, and counterstained with DAPI to label cell nuclei and FITC-phalloidin 488 (green) to label cytoskeleton. Cells were observed using confocal laser scanning microscopy (Zeiss LSM-710). To explore the mechanism of En-CNP internalization, serum-free medium containing one of the following inhibitors (or pure medium [Blank]) was added to cells: sodium azide (NaN3, 75 µM), chloropromazine (CPZ, 20 µM),24 dimethyl amiloride (DMA, 100 µM),25 or nystatin (NYS, 25 µM).24 After 1 h, FITC-miRNA-carrying En-CNP was added. After further incubation for 2 h, cells were analyzed as described above for the uptake assay. Each experiment was performed in triplicate. 2.8. Escape of Nanoparticles from Endosome/Lysosome 4T1 cells were seeded on commercial confocal microscopic dishes (1.0 × 105 cells/well) and incubated overnight, then treated for 1, 4 h with En-CNP loaded with FITC-miRNA (500 µL, equivalent to 2 µg miR-34a). Cells were washed three times with cold PBS containing heparin (20 U/mL) and once with serum-free medium, then incubated for 1 h with Lyso-tracker (Beyotime, China). Samples were washed in PBS and observed using a Leica TCS SP5 AOBS confocal microscope (Leica, Germany). 2.9. Wound-Healing Assay Migration and mobility of 4T1 cells were examined in an in vitro wound-healing assay. Cultured cells were seeded into a 12-well plate and grown to 80% confluence, then culture medium was collected. The scratch wounds were created in confluent monolayers of 4T1 cells using a sterile, 10-µL pipette tip. After washing away suspended cells, 500 µL of collected medium containing CP2K, En-CNP or En-unCNP was added (equivalent to 2 µg miR-34a). After 4-h incubation, the culture medium was replaced with complete fresh medium. Cell migration into the wound space was estimated at 24 h after wounding by measuring the cleared space between the wound edges in images captured using a Zeiss Axiovert 40 inverted microscope (Germany). 2.10.

In Vitro Anti-Tumor Activity Assay and Gene Silencing Assay 6


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We transfected cells for 4 h with naked miR-34a or three nanoparticle formulations (100 µL, equivalent to 0.5 µg miR-34a) in the presence of collected cultured medium, then the medium was replaced with complete medium and the cells were incubated another 72 h. The ability of the different treatments to inhibit 4T1 proliferation was assessed using the MTT assay. Untreated cells served as controls and were defined as showing 100% viability. To measure transcription of the histone deacetylase 1 (HDAC1) gene in vitro, cells were treated for 4 h with miR-34a-carrying nanoparticles in 500 µL of collected cultured medium (equivalent to 2 µg miR-34a, n=3 cultures per condition) and incubated another 24 h in complete fresh medium, then total RNA was extracted using an RNA isolation kit (TianGen, China) and reverse-transcribed into cDNA using the TIANscript RT kit and the HDAC1 primers 5’-GAACTACCCACTGCGAGACG-3’ (sense) and 5’-ACAGGGAATCTGAGCCACAC-3’ (antisense). Levels of HDAC1 mRNA were analyzed using the SsoFast EvaGreen Supermix and iCycler iQ 5 system (BioRad, USA) with β-actin as internal control. 2.11. Immunogenicity Assay Female Balb/c mice aged 6-8 weeks were obtained from the Laboratory Animal Center of Sichuan University (Chengdu, China). Animal studies were approved by the Institutional Animal Care and Ethics Committee of Sichuan University. Mice were intravenously injected with naked miR-34a or nanoparticles complexed with miR-34a (10 µg miR-34a per mouse, n = 5 per treatment). Mice injected intravenously with lipopolysaccharide (LPS; Sigma; 30 µg per mouse) served as a positive control. At 4 h after injection, serum was collected and assayed for IL-6 and IL-12 using an enzyme-linked immunosorption assay (ELISA) kit (BD science, USA) according to the manufacturer’s instructions. Absorbance at 450 nm was recorded. At 24 h after injection, mice were sacrificed and the major organs such as liver, kidney and lung were collected, fixed in 4% paraformaldehyde and embedded in paraffin. Sections were treated with hematoxylin and eosin (H&E) staining and examined by light microscopy (Zeiss Axiovert 40). 2.12. Biodistribution of Nanoparticles Mice were injected with 4T1 tumor cells (5×105 cells per mouse, 200 µL), and on approximately day 14, when the tumors were palpable, the in vivo fluorescence imaging and biodistribution experiments were performed. Animals were injected intravenously with nanoparticles loaded with CY5-labeled miRNA (200 µL nanoparticles, 10 µg miR-34a per mouse). After 2 h, mice were sacrificed, organs were collected, and fluorescence images of organs were obtained using the IVIS Spectrum system (Caliper Life Sciences, USA). Then tumors were cryopreserved in tissue-freezing medium (Leica) and cut into 8-µm slices using a microtome (Leica CM1950). Slices were stained with DAPI to label the nucleus, then examined for fluorescence using a Zeiss LSM-710 confocal laser scanning microscope. 2.13.

In Vivo Anti-Tumor Efficacy 7



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We subcutaneously injected 4T1 cells (5×105, 200 µL) into the right flank of Balb/c female mice, and on day 7, mice were randomly divided into 5 groups that were injected intravenously with PBS, naked miR-34a, CP2K, En-CNP or En-unCNP on days 7, 9, 11, and 13 after initial injection with tumor cells (n = 10 animals per group). Each injection contained 10 µg miR-34a per mouse. Changes in tumor growth and body weight were monitored; volume was calculated using the formula (L×S2/2), where S refers to the shortest diameter and L to the longest. The tumor inhibition ratio (TIR) was calculated using the equation TIR(%) = (1-Vt/Vc)× 100%, where Vt and Vc refer to the average tumor volume on day 22 in the treatment and control groups, respectively. On day 22, animals were sacrificed and tumors were collected and used to prepare paraffin-embedded sections that were stained with H&E staining described in section 2.11. To assess the degree of apoptosis, paraffin-embedded tumor tissue was subjected to the transferase-mediated dUTP nick end-labeling (TUNEL) assay according to the manufacturer's instructions (QIA33, Merck, Germany). To evaluate inhibition of tumor cell proliferation, sections were assayed for Ki-67 antigen according to the manufacturer’s protocol (RM-9106-S0, Thermo Scientific, USA) and examined under a microscope (Axiovert 40CFL, Germany). When the longest tumor diameter exceeded 20 mm, mice were sacrificed and the overall survival test was carried out as described in anti-tumor growth part. 2.14. Statistical Analysis Experiments were performed at least in triplicate. Data are shown as the mean ±SD. Differences were assessed for significance using one-way analysis of variance (ANOVA) and Student’s t test. P < 0.05 was considered to be significant.

3. RESULTS 3.1. Synthesis and Characterization of CD-PEI2K, CP-C-PEG, and CP-unC-PEG β-cyclodextrin-PEI2K (CD-PEI2K) and CD-PEI2K-MMP2-cleavable-peptidePEG (CP-C-PEG) were synthesized as shown in Figure 1A. The chemical structures and molecular weight of CD-PEI2K were determined by 1H NMR (Figure 1B) and gel permeation chromatography (Figure S1A), respectively. The 1H NMR of CP-C-PEG was showed in Figure 1B. Hydrogen atom signals were observed for β-cyclodextrin (5.1 ppm), PEI (2.8-3.2 ppm) and PEG (3.6-3.8 ppm), confirming that the product was CP-C-PEG. The molecular composition of CP-C-PEG was determined based on comparisons of the integrated peak areas for the H protons of the CD rings with the corresponding integrated peak areas of PEI and PEG. The empirical molar ratio of PEG2K-MMP2-cleavable peptide: PEI2K: CD was 1.29:1:0.37. As a control for the effects of MMP2 cleavage of the peptide, we prepared CD-PEI2K-MMP2-uncleavable peptide-PEG (CP-unC-PEG) using the same method described above, and the molar ratio of PEG2K-MMP2-uncleavable peptide: PEI2K: CD was 1.21:1:0.37. 8


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3.2. Gelatin Assay We measured MMP2 secretion by various cell lines that are typically used as in vitro models of triple-negative breast cancer in order to identify one suitable for our studies. Gelatin zymography was used to assess levels of secretion.26 Human fibrosarcoma cell line HT1080 secreted a high level of MMP2, and the adenocarcinoma cell line 4T1 showed high secretion of both active MMP2 and inactive precursor pro-MMP2 (Figure 1C). ELISA to quantify MMP2 secretion showed 19.78±2.71 ng/mL in the medium of 4T1 cultures and 25.81±2.37 ng per mg of total protein in xenograft 4T1 breast tumors in mice. These results identified 4T1 as expressing suitably high levels of MMP2 for our studies.

Figure 1. A. Schematic of the synthesis of experimental materials. B. NMR analysis of CD-PEI2K, CP-C-PEG and CP-unC-PEG. C. Gelatin zymography assay of MMP2 expression by 4T1 cells.

3.3. Physical Properties of Nanoparticles Dynamic light scattering showed average hydrodynamic diameter to be 54.83±0.42 nm for CP2K (CD-PEI2K/miRNA), 99.17±1.54 nm for En-CNP (CP-C-PEG/miRNA) and 105.53±2.21 nm for En-unCNP (CP-unC-PEG/miRNA) (Figure 2A). Particles of En-CNP and En-unCNP were slightly larger due to conjugation of peptide-PEG2K. Nevertheless, all these nanoparticles were monodisperse, based on their polydispersity index < 0.3. Consistent with these results, transmission electron microscopy showed all nanoparticles to be uniformly spherical and monodisperse (Figure 2C). As expected, zeta-potential was lower for En-CNP (14.10±0.85 mV) and En-unCNP (15.57±1.50 mV) than for CP2K nanoparticles (22.83±0.31 mV), reflecting shielding by the PEG coating (Figure 2B). Such positive surface charge contributed to the good miRNA binding capacity of our cationic gene vectors. To assess the stability of nanoparticles, size was monitored over four days at room temperature, starting on the day of preparation. No significant changes were 9



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observed (Figure 2D). Since a PEG layer can prevent nanoparticles from interacting nonspecifically with serum components and thereby protect them in the systemic circulation,27-31 we incubated the nanoparticles with mouse serum and monitored their size at 2 h. While cationic carriers such as PEI25K and CP2K were unstable during incubation, PEGylated En-CNP and En-unCNP showed significantly lower interaction with blood proteins (Figure 3A). These results suggest that the PEG layer can enhance the stability of En-CNP nanoparticles in the presence of serum proteins.

Figure 2. A. Size distribution of CP2K, En-CNP and En-unCNP nanoparticles, as measured by dynamic light scattering. B. Zeta-potential of nanoparticles. Results were shown as mean±SD (n = 3). C. Transmission electron micrographs of CP2K, En-CNP and En-unCNP nanoparticles. D. Hydrodynamic nanoparticle size and stability over four days, starting on the day of preparation (day 0). Results were shown as mean±SD (n = 3).

3.4. Cytotoxicity of Synthetic Materials CP2K, CP-C-PEG and CP-unC-PEG Cytotoxicity of different polymers on 4T1 cells after 24-h incubation was assessed using the MTT assay. CP2K, CP-C-PEG and CP-unC-PEG all showed minimal cytotoxicity at concentrations below 12 µg/mL (Figure 3B).This suggests that our concentration 8 µg/mL in subsequent in vitro experiments can be considered to cause negligible cytotoxicity. Nevertheless, PEI25K showed significant cytotoxicity in all the investigated concentrations. Therefore, we did not choose PEI25K as the positive control in the following experiments.

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Figure 3. A. Stability of nanoparticles after incubation in mouse serum for 2 h at 37 °C. B. Viability of 4T1 cells treated with different concentrations of CD-PEI2K, CP-C-PEG and CP-unC-PEG. C-D. Quantitation of uptake of CP2K, En-CNP, and En-unCNP by 4T1 cells. Results were shown as mean±SD (n = 3). ***P < 0.001.

3.5. Nanoparticle Uptake In Vitro Although PEGylation can improve the stability of nanoparticles, it can also hinder their internalization. We hypothesized that removal of the PEG layer from En-CNP as a result of peptide cleavage by MMP2 in the tumor microenvironment would re-expose the positive nanoparticle surface and thereby enhance uptake of nanoparticles. Consistent with this hypothesis, we found that cells incubated with En-CNP showed much stronger intracellular fluorescence than did cells incubated with En-unCNP, based on fluorescence-positive cell sorting (Figure 3C-D). Internalization of nanoparticles was also investigated using confocal laser scanning microscopy. Treating 4T1 cells with the cationic materials CP2K and En-CNP led to efficient internalization of CY5-miRNA (red) into cells, where it distributed throughout the cytoplasm. The intensity of internalized fluorescence increased with longer incubation. In contrast, treating cells with En-unCNP did not lead to extensive intracellular red fluorescence in the cytoplasm (Figure 4A-B). Since three-dimensional tumor models may better resemble many aspects of intratumor pathophysiology,32 we also examined nanoparticle internalization in multicellular spheroids. Internalized fluorescence intensity was greater for CP2K and En-CNP than 11



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for En-unCNP (Figure 4C-D). To investigate the potential endocytotic pathway(s) of En-CNP, uptake experiments were performed in the presence of various inhibitors, each of which blocked a single endocytosis pathway. Chloropromazine markedly decreased the uptake efficiency of En-CNP, reducing it to 70% of that in the control group (Figure 5A). Chlorpromazine can disrupt clathrin-membrane interactions, inhibiting clathrin-dependent endocytosis. Nystatin, an inhibitor of caveolae-mediated endocytosis, reduced uptake of En-CNP to 80% of the blank level, while the metabolic inhibitor (sodium azide) 33 reduced it to 75% (P < 0.05). These results suggest that uptake of En-CNP by cells occurs via energy-dependent endocytosis mediated by clathrin and caveolae.

Figure 4. A-B. Confocal micrographs of 4T1 cells incubated at 37 °C with nanoparticles for (A) 2 h and (B) 4 h. Red indicates CY5-miRNA; green, FITC-phalloidin; and blue, nuclei. C-D. Confocal micrographs of multicellular 4T1 spheroids incubated at 37 °C with nanoparticles for (C) 2 h and (D) 12 h.

3.6. Escape of MiR-34a Cargo from Endosome/Lysosome For therapeutic efficacy, the miRNA cargo of our nanoparticles must escape the endosome/lysosome after particle internalization. We wanted to test this directly, particularly since coating nanoparticles with PEG has been shown to hinder the escape of payload from endosome/lysosome into the cytoplasm.34-36 Confocal laser scanning microscopy of 4T1 cells that had been incubated for 1 h with En-CNP showed that most FITC-miRNA (green) co-localized with Lyso-tracker (red), which 12


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labels endosome/lysosome, giving rise to yellow fluorescence (Figure 5B). After a total of 4 h incubation, most green fluorescence was observed in the cytoplasm, with much less miRNA overlapped with red fluorescence. These results indicate that the payload in En-CNP can escape from endosome/lysosome after being internalized by cells.

Figure 5. A. Effects of endocytosis inhibitors on uptake of En-CNP by 4T1 cells. Results were shown as mean±SD (n = 3). *P

PEGylated Cationic Vectors Containing a Protease-Sensitive Peptide

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