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Receptor-Mediated Surface Charge Inversion Platform Based on Porous Silicon Nanoparticles for Efficient Cancer Cell Recognition and Combination Therapy Feng Zhang, Alexandra Correia, Ermei M. Mäkilä, Wei Li, Jarno J. Salonen, Jouni Hirvonen, Hongbo Zhang, and Hélder A. Santos ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b02196 • Publication Date (Web): 01 Mar 2017 Downloaded from http://pubs.acs.org on March 1, 2017

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ACS Applied Materials & Interfaces

Receptor-Mediated

Surface

Charge

Inversion

Platform Based on Porous Silicon Nanoparticles for Efficient Cancer Cell Recognition and Combination Therapy

Feng Zhang a, Alexandra Correia a, Ermei Mäkilä b, Wei Li a, Jarno Salonen b, Jouni J. Hirvonen a, Hongbo Zhang a, 1,*, Hélder A. Santos a,*

a

Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of

Helsinki, FI-00014 Helsinki, Finland b

Laboratory of Industrial Physics, Department of Physics and Astronomy, University of

Turku, FI-20014 Turku, Finland 1

Current Affiliation: Department of Pharmaceutical Sciences Laboratory, Åbo Akademi

University, FI-20520 Turku, Finland

*Corresponding authors. Emails [email protected] (H.Z.); [email protected] (H.A.S.). Tel.: +358-2941-59661 KEYWORDS: drug delivery; surface charge inversion; selective cell recognition; targeting; combination therapy 1 ACS Paragon Plus Environment


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ABSTRACT Negatively charged surface-modified drug delivery systems are promising for in vivo applications as they have more tendency to accumulate in tumor tissues. However, the inefficient cell uptake of these systems restrict their final therapeutic performance. Here, we have fabricated a receptor-mediated surface charge inversion nanoparticle made of undecylenic acid modified, thermally hydrocarbonized porous silicon (UnTHCPSi) nanoparticles core and sequentially modified with polyethylenimine (PEI), methotrexate (MTX) and DNA aptamer AS1411 (herein termed as UnTHCPSi -PEI-MTX@AS1411) for enhancing the cell uptake of nucleolin-positive cells. The efficient interaction of AS1411 and the relevant receptor nucleolin caused the disintegration of the negative-charged AS1411 surface. The subsequent surface charge inversion and exposure of the active targeting ligand, MTX, enhanced the cell uptake of the nanoparticles. Based on this synergistic effect, the UnTHCPSi -PEI-MTX@AS1411 (hydrodynamic diameter is 242 nm) were efficiently internalized by nucleolin-positive MDA-MB-231 breast cancer cells, with an efficiency around 5.8 times higher than that of nucleolin-negative cells (NIH 3T3 fibroblasts). The receptor competition assay demonstrated that the major mechanism (more than half) of the internalized nanoparticles in MDA-MB-231 cells were due to the receptor-mediated surface charge inversion process. Finally, after loading of sorafenib, the nanosystem showed efficient performance for combination therapy with an inhibition ratio of 35.6%.

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Introduction Charge effects can significantly determine the in vivo behavior of nanoparticles (NPs).1-3 Generally, compared with positively charged NPs, neutrally or negatively charged NPs own better biocompatibility, reduced protein surface adsorption and antigenicity, and longer circulation half-lives.4-7 These advantages can translate into efficient NPs accumulation in the tumor tissue by the enhanced permeability and retention effect.8-9 However, the inefficient cell internalization of neutrally or negatively charged NPs may cause reduced therapeutic performance.10 To overcome the opposite effects during in vivo transport and cell internalization of therapeutic NPs, charge inversion surface has been developed as an efficient strategy.11 In view of the different pH values or enzyme concentrations in the tumor tissue, most of the efforts have been put in the construction of pH-triggered12-14 or enzyme-degradable surface charge inversion systems.15-16 However, the response to the subtle pH differences (from pH 7.4 to pH 6.8) or inefficient enzyme effects has always led to long response times (usually, more than 24 h).17-18 Here, we propose a simple and efficient strategy to prepare a negatively charged inversion surface NP based on the receptor-mediated surface charge inversion (RMSCI). The 26-mer G-rich DNA aptamer AS1411 was selected as the anionic component to construct the charge inversion surface, because it can recognize the receptor protein nucleolin,19 which is highly expressed on the surface of most malignant cells, but very limitedly expressed on normal cells.20-21 This way, a stable AS1411-nucleolin complex with high protein-binding affinity can be formed.21-24 The sequential conjugation of polyethylenimine (PEI) and methotrexate 3 ACS Paragon Plus Environment


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(MTX) on undecylenic acid modified porous silicon (UnTHCPSi) NPs will build up a positively charged nanoparticle. And the subsequent absorption of AS1411 with maximum payload will form the negatively charged surface. Our hypothesis for RMSCI process is based on the resistive forces, such as membrane wrapping and receptor diffusion during NPs internalization,25-26 which could also be the driven forces for releasing AS1411 from UnTHCPSi-PEI-MTX@AS1411. In addition, as the maximum DNA payload in this system, the weak electrostatic interaction between external AS1411 and PEI will set stage for the release of AS1411 from NPs. As the result, the subsequent surface charge inversion and exposure of targeting moiety (MTX) will facilitate cell uptake process. In view of the overexpressed nuleolin on membrane of nuleolin-possitive cells (e.g., MDA-MB-231 cells), the relevant RMSCI process will be more efficient than that of nucleolin-negative cells (e.g., NIH 3T3 fibroblasts). In this UnTHCPSi-PEI-MTX@AS1411 NPs based drug delivery system, both AS1411 and MTX have anticancer properties. The AS1411 can inhibit the function of nucleolin and result in oncogenic effects and proliferation of cancer cells.21, 24 MTX can inhibit the function of dihydrofolate reductase and thymidylate synthetase, blocking the synthesis process of dihydrofolate and deoxythymidine monophosphate.27-28 After loaded with the tyrosine kinase inhibitor sorafenib (SFN) drug,29 the whole system was also used for cancer combination therapy. We demonstrated the selective cell recognition capacity by confocal fluorescence imaging and flow cytometry, and the induced selective cytotoxicity was tested by a cell viability assay. The relevant uptake mechanism was further confirmed by the intracellular behavior of the NPs traced by double fluorescent-labeled UnTHCPSi-PEI-MTX@AS1411 4 ACS Paragon Plus Environment

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and receptor competition assay. Finally, the performance of combination therapy was tested using the sorafenib-loaded UnTHCPSi-PEI-MTX@AS1411 and MDA-MB-231 breast cancer cells.

RESULTS AND DISCUSSION Design, Preparation and Characterization of UnTHCPSi-PEI-MTX@AS1411 NPs. As a classic gene transfection reagent,30-31 polyethylenimine (PEI) was selected as a model cationic polymer to load AS1411 aptamer. MTX was used for post-modification on the PEI’s surface. Firstly, the polycation-induced cell membrane disruption of PEI can be reduced by blocking the amine groups of PEI. 32-33 Furthermore, the covalently conjugated MTX through amide bond can maintain its targeting capacity and anticancer activity.34 In this view, the endowed dual recognition system (AS1411 and MTX to target the nucleolin and folic acid receptor of the cells, respectively) may have a synergetic effect in the process of RMSCI in order to enhance the selective cell recognition capacity. In addition, the hydrogen bond acceptor ability of MTX is also necessary to maintain the positive charge of the system,35 which can guarantee the payload of AS1411 to shield the system with high surface density. Finally, the UnTHCPSi NPs were used as the core framework of the drug delivery system, because of their good biocompatibility and porous structure for SFN loading.35-38 Briefly, the UnTHCPSi NPs were prepared by electrochemical etching and wet-milling procedure,

as

described

elsewhere.39-40

Carbodiimide

chemistry

based

on

N-

hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) was used for the follow-up PEI conjugation and MTX modification of the UnTHCPSi NPs.41 5 ACS Paragon Plus Environment


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Then, the poorly water-soluble antiangiogenic drug SFN was loaded into the PSi pores.38 In order to promote the RMSCI process, AS1411 was adsorbed by electrostatic interactions onto the surface of UnTHCPSi-PEI-MTX instead of conjugated to the surface. Unlike the traditional DNA/PEI complex that require high PEI nitrogen atoms to DNA phosphate ratio to maintain the positive charge of the complex to facilitate the cell endocytosis42, we used the full loading capacity to carry DNA aptamer to maximize the negative charge-depended safety and, most importantly, to achieve the best performance of the RMSCI. The fabrication process is shown in Fig. 1A.

Figure 1. (A). The fabrication process of drug delivery system UnTHCPSi-PEIMTX@AS1411 and SFN-loaded UnTHCPSi-PEI-MTX@AS1411 NPs. (B). Size and


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relevant PDI of the prepared NPs. (C). ζ-potential of the prepared NPs. (D). TEM images of the obtained NPs.

Dynamic light scattering showed the changes of the size distribution of the different types of NPs prepared (Fig. 1B). Briefly, after PEI conjugation, the average hydrodynamic diameter increased ca. 30 nm, but the PdI decreased. The ζ-potential increased from −30 mV to 40 mV (Fig. 1C). After the conjugation of MTX, the size of the UnTHCPSi increased around 10 nm, which might be attributed to the property of the MTX molecules that differs from PEI. The ζpotential decreased by ~4−5 mV. No obvious change in either size or ζ-potential was observed after the loading of SFN, indicating that SFN was mainly adsorbed inside the UnTHCPSi NPs. After DNA aptamers adsorption onto the surface of the modified NPs, the PdI increased slightly, but still showing good dispersion stability of the NPs. Transmission electron microscopy (TEM) images showed the structures of different types of NPs prepared (Fig. 1D and Fig. S1). The successful synthesis processes were also confirmed by Fourier transform infrared spectroscopy (FTIR) (Fig. S2). Colloid Stability. To investigate the colloidal stability of the UnTHCPSi-PEIMTX@AS1411, Milli-Q water (pH 6.0), HBSS−HEPES buffer (pH 7.4) and 10% FBS buffer (pH 7.4) were used (Fig. S3). Comparing to Milli-Q water, the NPs were less stable in the HBSS-HEPES buffer solution. However, UnTHCPSi-PEI-MTX@AS1411 NPs were stable (400 - 500 nm) in FBS buffer (pH 7.4). Drug Loading Degree. Different release methods were used to determine the loading degree (LD) of the three therapeutic components added to the UnTHCPSi NPs. The 7 ACS Paragon Plus Environment


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immersion method was used to release SFN, and base-catalyzed amide bond cleavage method was adopted to release MTX. LD of SFN (LDSFN) and MTX (LDMTX) were determined by high-performance liquid chromatography (HPLC, see in the Materials and Methods section). The LDSFN was 30 ± 2% and LDMTX was 8 ± 0%. In addition, the difference of LDSFN between SFN-loaded UnTHCPSi-PEI-MTX and SFN-loaded UnTHCPSi-PEIMTX@AS1411 NPs was also investigated to check whether the electrostatic absorption of AS1411 influenced on the loading of SFN into UnTHCPSi NPs. The HPLC data showed the LDSFN of SFN-loaded UnTHCPSi-PEI-MTX@AS1411 NPs was around 1% less than that of SFN-loaded UnTHCPSi-PEI-MTX NPs, indicating that AS1411 adsorption onto the NPs did not interfere significantly with the loading of SFN. For AS1411, the LD was calculated by UV-Vis, as described in Supporting Information and in Table S1. The LDAS1411 in Milli-Q water and HBSS−HEPES buffer (pH 7.4) were 33 ± 1% and 26 ± 1%, respectively. AS1411

Payload-Depended

Surface

Charge

Inversion

of

UnTHCPSi-PEI-

MTX@AS1411 NPs. The surface charge of UnTHCPSi-PEI-MTX@AS1411 NPs was adjusted by the payload of AS1411, and this data can also reflect the charge inversion tendency of the NPs followed by the release of AS1411. The different payloads of AS1411 were achieved by adjusting the mass ratio of UnTHCPSi NPs to AS1411 (NP: DNA), and the AS1411 payload-dependence surface charge inversion relationship was investigated by measuring the ζ-potential in Milli-Q water (pH 6.0) and HBSS−HEPES buffer (pH 7.4) independently (Fig. 2). By increasing the amount of AS1411, a PEI-AS1411 complex (AS1411@PEI) will be firstly formed on the surface of the particle, and an AS1411 monolayer (PEI@AS1411) will 8 ACS Paragon Plus Environment

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be finally created to coat the whole NP. For cell uptake, the charge inversion is referred to as a process but not an outcome. The increasing of the surface charge potential will facilitate the cell uptake process of the NPs. In view of the cell recognition capacity, here we used the maximum loading degree of AS1411 to make sure that the uptake efficiency of normal cells to the UnTHCPSi-PEI-MTX@AS1411 NPs could be mostly suppressed. The relevant analysis is shown in Table S2.

Figure 2. Surface charge AS1411 dependency inversion tendency in HBSS−HEPES buffer (pH 7.4) (A) and in Milli-Q water (pH 6.0) (B).

Drug Release of SFN and MTX. The SFN release curves from UnTHCPSi-PEI-MTX and UnTHCPSi-PEI-MTX@AS1411 NPs, are shown in Fig. S4. No obvious release of SFN was observed by HPLC from the UnTHCPSi NPs in HBSS−HEPES buffer (pH 7.4). In contrast, SFN released very fast in 10% FBS buffer, due to the strong affinity of SFN to the proteins in FBS buffer,38 with most of the loaded SFN (around 90%) being released within 10 min. However, after adsorption with AS1411, the SFN release tendency was less than 45%



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within 6 h, indicating that the UnTHCPSi -PEI-MTX@AS1411 NPs can more efficiently sustain the SFN release than the UnTHCPSi-PEI-MTX. No MTX release was observed in HBSS−HEPES buffer at pH 7.4 with or without 3% protease due to the stable amide bond in weak acid conditions. However, the conjugated MTX can still maintain the targeting capacity and anticancer activity.34 Investigation

of

Selective

Cell

Recognition

Capacity

of

UnTHCPSi-PEI-

MTX@AS1411 NPs. Firstly, confocal fluorescence imaging was used for evaluating the cell internalization efficiency of UnTHCPSi-PEI-MTX@AS1411 NPs. UnTHCPSi-PEI-MTX NPs with a positively charged targeting moiety, PEI-MTX, were used as positive control. Simultaneously, a normal DNA (deoxyribonucleic acid sodium salt from calf thymus) coated system, UnTHCPSi-PEI-MTX@Normal DNA was introduced as negative control, because this kind of double-strand DNA does not have specific interaction with cell membrane, and only acts as the anionic moiety for the negatively charged surface. All the NPs were conjugated with fluorescein isothiocyanate (FITC) and the cells were stained with CellMask™ Deep Red. The obtained confocal images are shown in Fig. 3A. After 3 h treatment, for UnTHCPSi-PEI-MTX NPs, both the cell membrane attachment and internalization were obvious in both cell lines, and the tendency was more pronounced after 12 h treatment. This indicates that the positively charged system had limited selectivity in cell targeting. For UnTHCPSi-PEI-MTX@Normal DNA NPs, the NP cell internalization in both cell lines was very low after 3 h treatment, and no increase on the cell NP uptake was observed after 12 h, with most of the NPs found around the cells. This confirms the very inefficient cells internalization of the negatively charged NPs. Similarly, inefficient cell 10 ACS Paragon Plus Environment

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uptake was also observed for UnTHCPSi-PEI-MTX@AS1411 NPs treated 3T3 fibroblasts. However, the FITC fluorescence was observed in the MDA-MB-231 cells treated with UnTHCPSi-PEI-MTX@AS1411 NPs for 3 h, which increased further after 12 h treatment.



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Figure 3. (A) Confocal fluorescence microscope images of 3T3 fibroblast and MDA-MB231 cells treated with FTIC-labeled NPs (red: cell membranes stained with CellMaskTM 12 ACS Paragon Plus Environment

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DeepRed; green: FITC-labeled NPs; yellow: co-localization of the NPs and the cell membrane). (B) Flow cytometry quantitative analysis of 3T3 fibroblast and MDA-MB-231 cells. The cells were incubated with the NPs for 3 h and 12 h at 37 °C. The table summarizes the mean fluorescence intensity for each sample. Data are shown as mean ± s.d. (n = 3).

Next, flow cytometry was used to evaluate the cell uptake efficiency of UnTHCPSi-PEIMTX@Normal DNA and UnTHCPSi-PEI-MTX@AS1411 NPs in 3T3 fibroblast and MDAMB-231 cells (Fig. 3B). All the tested cell samples were treated with trypan blue to quench the outer fluorescence signal coming from the adsorbed NPs to the cells’ surface. For MDAMB-231 cells, UnTHCPSi-PEI-MTX@AS1411 NPs showed around 1.6 and 4.7 times higher uptake than that of UnTHCPSi-PEI-MTX@Normal DNA NPs after 3 h and 12 h treatment, respectively. For 3T3 fibroblasts, the cell uptake difference between both NPs was not so pronounced. This was expected since these cells express less nucleolin than MDA-MB-231 cancer cells, 43 and thus, the fluorescence intensity of UnTHCPSi-PEI-MTX@AS1411 NPs treated MDAMB-231 cells was around 2.7 and 5.8 times of UnTHCPSi-PEI-MTX@AS1411 treated 3T3 fibroblasts after 3 and 12 h treatment. The different cells uptake efficiency of UnTHCPSi-PEI-MTX@AS1411 NPs for MDAMB-231 cells and 3T3 fibroblasts were also demonstrated by comparing with the positive control (UnTHCPSi-PEI-MTX). For the MDA-MB-231 cells, the cell uptake of Un-THCPSiPEI-MTX NPs was around 2.5 times higher than that of UnTHCPSi-PEI-MTX@AS1411 NPs after 3 h treatment, but reduced to 1.5 times after 12 h. This indicates the enhanced cell 13 ACS Paragon Plus Environment


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uptake efficiency of UnTHCPSi-PEI-MTX@AS1411 NPs during prolonged incubation time. For 3T3 fibroblasts, the tendency was the opposite of the MDA-MB-231 cells. The fluorescence intensity of UnTHCPSi-PEI-MTX NPs was 4.4 times higher than that of UnTHCPSi-PEI-MTX@AS1411 NPs after 3 h treatment, and increased to 5.2 times after 12 h. (the methods and equation are described in the Materials and Methods section). Cytocompatibility and Cell Recognition-Dependent Selective Cytotoxicity of the NPs. As the RMSCI system was constructed by PEI, MTX and AS1411, it could induce cell toxicity. Thus, the selective targeting capacity of UnTHCPSi-PEI-MTX@AS1411 NPs with selective cytotoxicity was also tested (Fig. 4). Comparatively, although UnTHCPSi showed good cytocompatibility after being conjugated with PEI for short incubation times, an increase of cytotoxicity was observed within 3 h, indicating that the toxicity of UnTHCPSiPEI NPs is mainly due to the cytotoxicity of PEI. After MTX modification of the UnTHCPSiPEI NPs, the cell viabilities were improved within 6 h, indicating that MTX conjugation reduced the cytotoxicity of UnTHCPSi-PEI NPs, in a time- and concentration-dependent manner. The reason might be attributed to the remained tertiary amine in PEI32, which cannot react with MTX, thus still causing membrane injury, in addition to the cytotoxicity of MTX alone. However, after being coated with normal DNA strands, the viability of both cell lines was increased, suggesting that the negatively charged surface of the NPs could efficiently shield the cytotoxicity of UnTHCPSi-PEI-MTX NPs by preventing strong interactions with the cells. After the treatment with UnTHCPSi-PEI-MTX@AS1411 NPs, the cytotoxicity of 3T3 fibroblasts was similar to that of UnTHCPSi-PEI-MTX@Normal DNA NPs over time. 14 ACS Paragon Plus Environment

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However, in MDA-MB-231 cells, the difference between the viability of UnTHCPSi-PEIMTX@Normal DNA NPs and UnTHCPSi-PEI-MTX@AS1411 NPs increased over the treatment time. The significant cytotoxicity of UnTHCPSi-PEI-MTX@AS1411 NPs in MDA-MB-231 cells after 24 h suggests that the surface shielding effect by the negatively charged AS1411 surface was sharply reduced compared to the NPs modified with normal DNA. Taking into account the cytotoxicity and cell uptake studies, it can be concluded that the significant difference in cytotoxicity was due to an enhanced cell internalization of the NPs.

In

addition,

although

UnTHCPSi-PEI-MTX@AS1411

NPs

showed

good

cytocompatibility compared to UnTHCPSi-PEI-MTX within 6 h, the notable cytotoxicity at 24 h indicating that AS1411 could only act as a temporary shield for the NPs in the presence of nucleolin-positive cells.



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Figure 4. Cell viability of 3T3 fibroblasts (A) and MDA-MB-231 cells (B) exposed to the different NPs assessed by the CellTiter-Glo® Luminescent assay after 1 h, 3 h, 6 h and 24 h treatments. Three concentrations of NPs, 25, 50 and 100 µg/mL were tested. Statistical 16 ACS Paragon Plus Environment

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analyses were made by one-way analysis of variance (ANOVA) with Bonferroni post-test. The levels of significance were set at probabilities of *p < 0.05, **p < 0.01, and ***p < 0.001. Data are shown as mean ± s.d. (n = 3).

Intracellular Studies of UnTHCPSi-PEI-MTX@AS1411 NPs. To further investigate the interaction of UnTHCPSi-PEI-MTX@AS1411 NPs with cells, double fluorescent-labeled UnTHCPSi-PEI-MTX@AS1411 NPs were prepared, which included Atto 590-labeled AS1411 and FITC-labeled UnTHCPSi for the confocal fluorescence microscopy imaging (Fig. 5). To avoid the influence of AS1411−nucleolin interactions, we have labelled with Atto 590 on the 3’-terminus extended with two thymine deoxyribotide units (TT).23 The release of AS1411 from UnTHCPSi-PEI-MTX NPs would be expected to lead to changes in the merged fluorescence signal. Thus, the change in the fluorescence signal of UnTHCPSi-PEIMTX@AS1411 NPs on cell membrane could be directly related to the AS1411 release. In addition, the intracellular DNA aptamer release behavior could thus be also observed. In Fig. 5, the Atto 590-AS1411 fluorescence signal was observed for UnTHCPSi-PEIMTX@AS1411 NPs at 3 h, and the fluorescence intensity was enhanced for both Atto 590 and FITC after 12 h. However, obvious changes in the merged fluorescence signal were observed on the cell membrane (the enlarged pictures are shown in Fig. S5), and the reduced fluorescence intensity of AS1411 indicating that the AS1411 release could have occurred before the cell internalization of UnTHCPSi-PEI-MTX@AS1411 NPs. The presentative changes were marked by arrows and squares in Fig. S5. Green dots on cell surface could be



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observed, showing that the release of AS1411 is very efficient. In addition, notable intracellular red dots could also prove that the release of AS1411 from NPs.

Figure 5. Confocal fluorescence microscope images of MDA-MB-231 cells treated with UnTHCPSi-PEI-MTX@AS1411 NPs (white: cell membranes stained with CellMaskTM DeepRed; green: FITC-labeled NPs; red: Atto 590-labeled AS1411; yellow: co-localization of the FTIC-labeled NPs and Atto 590-labeled AS1411).


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Receptor Competition Assay. Finally, the receptor competition assay was used to evaluate the roles and possibilities of the different kinds of internalization mechanism of the NPs (Fig. 6). Three inhibitor competition groups, including folic acid (FA), AS1411 and FA+AS1411 were used to investigate the influence on cell internalization by UnTHCPSi-PEIMTX@AS1411 NPs in this competition assay. The data calculated based on the flow cytometry measurements, showed that the mean inhibition ratio of AS1411, FA and AS1411+FA (termed as InRAS1411, InRFA and InRAS1411+FA) was 73%, 61% and 81%, respectively (the methods and equation are shown in the Materials and Methods section, and the schematic diagram of the calculations is showed in Fig. S6). Firstly, for the AS1411+FA (InRAS1411+FA) of 81%, with exception for the possibility of incomplete inhibition, we can attribute the rest 19% to the effect of other nonspecific internalization pathways, such as pinocytosis and clathrin/caveolae -mediated endocytosis.4445

Since the sum of the inhibition effect of AS1411 and FA (InRAS1411+InRFA) was 134%, with

53%. higher than the InRAS1411+FA. This means that the AS1411-mediated internalization and FA receptor-mediated internalization processes are not independent for cell uptake of UnTHCPSi -PEI-MTX@AS1411 NPs. This data suggested the existence of RMSCI mechanism, as free AS1411 inhibitor could restrict AS1411 releasing from UnTHCPSi-PEIMTX@AS1411 surface and then prevent the subsequent exposing of MTX. Thus, the overlapped 53 pp inhibited internalization was RMSCI-based FA receptor-mediated endocytosis. Furthermore, comparing the InRAS1411 with InRAS1411+FA, it can be noticed that there are 8% increased inhibition ratio after adding FA, indicating that the independent FA receptor-mediated endocytosis is attributed to the whole internalization process. For 19 ACS Paragon Plus Environment


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InRAS1411, it suggests there is still 20% inhibition ratio caused by AS1411 inhibition after removing the overlapped part (InRAS1411+InRFAInRAS1411+FA). This 20% includes the effect of the nucleolin-mediated endocytosis (this pathway will be processed by the NPs conjugated by AS1411)46-47 and the nonspecific internalization triggered by electrostatic absorption of the NPs. Although we cannot distinguish the shares of these two parts by this competition assay, the ratio of the RMSCI mechanism in the cell internalization process took more than 50% (RMSCI-based FA receptor-mediated endocytosis). The aforementioned mechanism is probably resulting from resistive force during the cell uptake to NPs, where the particle endocytosis has been reported to enhanced by size.25, 48 Since the size of UnTHCPSi-PEIMTX@AS1411 NPs is 242 nm, it can be predicted that the RMCSR process is a result of the equilibrium of the resistive force and the internalization force. All the calculations above are based on the assumption that the single inhibitor will not affect other internalization pathways.

Figure 6. Flow cytometry quantitative analysis of MDA-MB-231 cells treated with particles (FITC-labeled UnTHCPSi-PEI-MTX@AS1411 NPs) and different inhibitors. The treatment 20 ACS Paragon Plus Environment

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time was 12 h at 37°C. All the cell samples were then quenched with trypan blue to remove the interference of the NPs adsorbed to the cells’ surface. The histogram summarizes the mean fluorescence intensity for each sample. The levels of significance were set at probabilities of *p < 0.05, **p < 0.01 and ***p < 0.001. Data are shown as mean ± s.d. (n = 3).

Demonstration of the RMSCI Mechanism. Based on the aforementioned results and analysis, we confirmed the existence of a RMSCI mechanism, as shown in Fig. 7 For 3T3 fibroblasts (Fig. 7A), as a result of the very low nucleolin expressed on the cell’s membrane surface (much less than cancer cells), reduced AS1411 escape from the UnTHCPSi-PEIMTX@AS1411 NPs will not lead to efficient charge inversion, and the whole nanosystem still maintained a negative charge, minimizing the NPs’ internalization by the cells. For MDA-MB-231 cells (Fig. 7B), the overexpressed nucleolin on cell membrane will lead to efficient interaction with AS1411 on the therapeutic NPs. However, as the existence of the resistive forces (membrane wrapping and receptor diffusion) during cell internalization and the electrostatic repulsion, the efficient cell endocytosis to the whole NPs will not efficient in the early stage. Thus, the continuous release of AS1411 from the UnTHCPSi-PEIMTX@AS1411 NPs will be the outcome of the equilibrium of the different forces and driven by the transport of nucleolin on the cell’s membrane, leading to the disintegration of the external layer of AS1411 and the surface charge inversion of the NPs. In addition to that, the exposure of the MTX segments will also facilitate the cell uptake to NPs by FA receptormediated endosytosis. In this RMSCI mechanism, although AS1411 did not directly 21 ACS Paragon Plus Environment


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participate in the process of cell internalization to the UnTHCPSi-PEI-MTX@AS1411 NPs, it acted as the cell recognition system for cancer cell targeting.

Figure 7. Predicted mechanism of the different responsive behavior of UnTHCPSi-PEIMTX@AS1411 NPs with the nucleolin-negative 3T3 fibroblasts (A) and nucleolin-positive MDA-MB-231 cancer cells (B). I. Limited AS1411 release from the UnTHCPSi-PEIMTX@AS1411 NPs by the low expressed nucleolin 3T3 fibroblasts; II. The negatively charged surface of UnTHCPSi-PEI-MTX@AS1411 NPs cause electrostatic repulsion, leading to an inefficient interaction with the cells; III. AS1411 recognizes the overexpressed nucleolin on the cancer cell’s surface, escaping from the UnTHCPSi-PEI-MTX@AS1411 NPs; IV. The MTX molecules of the UnTHCPSi-PEI-MTX NPs are exposed to the cells and 22 ACS Paragon Plus Environment

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trigger the FA-mediated endocytosis; V. The continuous release of AS1411 from the UnTHCPSi-PEI-MTX@AS1411 NPs results in the increase of the surface charge potential, causing a nonspecific electrostatic absorption between UnTHCPSi-PEI-MTX@AS1411 NPs and the cell membrane. IV and V are non-independent processes of each other.

Combination Therapy Performance of SFN-loaded UnTHCPSi-PEI-MTX@AS1411 NPs. The antiproliferation effect of the therapeutic particles (SFN-loaded UnTHCPSi-PEIMTX@AS1411 NPs) was investigated using MDA-MB-231 cells. In the cell growth inhibition study, the mixture of the three drugs (MTX+SFN+AS1411) showed better antiproliferation performance than any of the single components alone, indicating that these three drugs own good synergetic anticancer effect. By comparison, the therapeutic particles showed lower cytotoxicity at 6 h. This might be due to the limited particle internalization in a short period. However, the performance of SFN-loaded UnTHCPSi-PEI-MTX@AS1411 NPs at 24 h was close to that of the drug mixture. This can be explained by the fact that the particle internalization was accelerated by the RMSCI, and also the cytotoxicity of PEI in the therapeutic system could lead to a more efficient antiproliferation effect.



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Figure 8. Cancer cell growth inhibition by treatment of MDA-MB-231 cells with pure MTX, SFN, AS1411, the combination of the free drugs (MTX+SFN+AS1411) and therapeutic particles (SFN-loaded UnTHCPSi-PEI-MTX@AS1411 NPs). Three different concentrations of the NPs (5, 10 and 20 µg/mL) were tested. The concentrations of the free drugs were based on the relevant drug loading degrees in the NPs. For MTX, the three concentrations were 0.4, 0.8 and 1.6 µg/mL. For SFN, the concentrations were 1.5, 3.0 and 6.0 µg/mL. And for AS1411, the concentrations were 1.3, 2.6 and 5.2 µg/mL. The MBA-MB-231 cells were exposed to the NPs for 6 h and 24 h at 37 °C. The levels of significance were set at probabilities of *p < 0.05, **p < 0.01 and ***p < 0.001. Data are shown as mean ± s.d. (n = 3).

CONCLUSIONS In this work, we used the DNA aptamer AS1411 to construct a receptor (nucleolin) mediated surface charge inversion nanosystem for cancer therapy. The anionic property of AS1411 endowed the NPs with good and long-term biocompatibility for nucleolin-negative cells (usually normal cells). However, this negatively charged surface could be disintegrated in the 24 ACS Paragon Plus Environment

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presence of nucleolin-positive cells (usually cancer cells), and then exposed the MTXconjugated surface with rising surface charge potential for efficient cell uptake. Based on the AS1411 identification, specific cell interaction by MTX moiety and nonspecific cell interaction by PEI, this RMSCI process has good cancer cell selectivity and anticancer activity. The diversity of DNA/RNA aptamers will enrich the function of this kind of nanoplatform for targeting different receptors. The physiological properties, such as the plasma stability and cell recognition capacity, could be further enhanced by DNA copolymer and other advanced DNA structures.

MATERIALS AND METHODS Materials. All the chemical reagents were purchased from Sigma-Aldrich (Finland). Methotrexate was purchased from TCI (Japan). Sorafenib (SFN) was purchased from LC laboratories® (USA). AS1411 and Atto 590 labeled AS1411 were purchased from Biomers (Germany). Hank’s balanced salt solution (10×HBSS), Dulbecco's Modified Eagle's Medium (DMEM), Roswell Park Memorial Institute (RPMI), Dulbecco's phosphate buffer saline (10× PBS), fetal bovine serum (FBS), trypsin (2.5%), sodium pyruvate, nonessential amino acids (100×NEAA), L-glutamine (200 mM), penicillin-streptomycin (100×PEST) were all purchased from HyClone (USA). Versene was purchased from Life Technologies (USA). Trypan blue was purchased from MP Biomedicals (Germany). The MDA-MB-231 breast carcinoma cells and NIH 3T3 Fibroblasts were obtained from American Type Culture Collection.



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Preparation of Undecylenic Acid modified Thermally Hydrocarbonized Porous Silicon (UnTHCPSi) NPs. The preparation of UnTHCPSi NPs was done electrochemical anodization as described in detail elsewhere.39 Synthesis of UnTHCPSi-PEI-MTX@AS1411. Synthesis UnTHCPSi-NHS ester NPs. 1 mg UnTHCPSi NPs were activated by 10 µL of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and then reacted with 2 mg of N-hydroxysuccinimide (NHS) in 1 mL of anhydrous dimethylformamide (DMF) for 24 h. The obtained UnTHCPSi-NHS ester NPs were harvested by centrifugation (Sorvall RC 5B plus, Thermo Fisher Scientific, USA) at 13,000g for 5 min, washed three times with anhydrous DMF and dispersed in 1 mL anhydrous DMF. Synthesis UnTHCPSi -PEI NPs. 10 mg PEI were dissolved in 1 mL of anhydrous DMF. The suspension of UnTHCPSi-NHS ester NPs was treated first with ultrasonic wave and then added into the PEI solution under vigorous stirring. After 12 h, the obtained UnTHCPSi-PEI NPs were harvested by the aforementioned procedures. Synthesis of MTX-NHS ester, UnTHCPSi-PEI-MTX NPs and FITC-labeled UnTHCPSiPEI-MTX NPs. MTX-NHS ester was prepared using standard carbodiimide chemistry. Briefly, 30 mg MTX (0.066 mmol) was activated by 12.9 µL (0.0726 mmol) EDC and 8.0 mg NHS (0.0695 mmol) for 18 h. The successful reaction was tested by thin-layer chromatography analysis. The obtained solution was used by mixing directly with UnTHCPSi-PEI NPs without further purification. After 24 h, the NPs were harvested by centrifugation at 13,000g for 5 min, and then wash with DMF, PBS buffer and Milli-Q water (pH 6.0) twice. For FITC-labeled UnTHCPSi-PEI-MTX NPs, before mixed with MTX-NHS 26 ACS Paragon Plus Environment

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ester, FITC-NHS ester (purchased from Sigma-Aldrich) was added (the mass ratio of NPs:FITC-NHS ester = 100:0.5). Loading SFN and AS1411. The obtained UnTHCPSi-PEI-MTX NPs were first washed with anhydrous DMF and then dispersed in the SFN DMF solution with the SFN concentration of 30 mg/mL. After vigorous stirring for 2 h, the NPs were centrifuged at 13,000g for 5 min, and then washed twice with PBS buffer and Milli-Q water (pH 6.0). Then, the NPs were dispersed in 1× HBSS buffer and the added amount of AS1411 was calculated based on the relevant loading degree. Characterization of the NPs. The hydrodynamic size (z-average), polydispersity index (PdI) and zeta-potential (ζ-potential) distribution of the NPs was measured by Zetasizer Nano ZS (Malvern Instruments Ltd.). The relevant data was recorded as the average of three measurements. The structure of the fabricated NPs was characterized by transmission electron microscope (TEM) under an acceleration voltage of 120 kV. The NPs samples were prepared by depositing them onto carbon-coated copper grids (300 mesh; Electron Microscopy Sciences, USA) and contrasting with 2% uranyl acetate solution. The NPs coated grids were dried at room temperature before the TEM imaging. The surface chemical modifications were characterized using Fourier transform infrared spectroscopy (FTIR; Bruker Vertex 70). The KBr pellets were processed by mixing 300 µg of the samples with 200 mg of KBr (spectroscopy grade, Sigma-Aldrich, Finland). FTIR spectra were recorded from 3600 to 650 cm-1 with a resolution of 4 cm-1 at room temperature using OPUS 5.5 software. 27 ACS Paragon Plus Environment


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Colloid

stability

of

UnTHCPSi-PEI-MTX@AS1411

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NPs.

After

treated

by

ultrasonication, 20 µg of the UnTHCPSi-PEI-MTX@AS1411 NPs were added into 1 mL of the relevant solution (Milli-Q water (pH 6.0), HBSS−HEPES buffer (pH 7.4) and 10% FBS buffer (pH 7.4)) and then stirred. The relevant size and PdI data was measured by Zetasizer Nano ZS at different time points. Drug Loading. Loading degree of SFN was tested by an immersion method. 20 µg of SFN-loaded UnTHCPSi-PEI-MTX@AS1411 NPs were suspended in 1 mL DMF and stirred for 2 h. Then the supernatant was obtained after centrifugation and the concentration of SFN was determined by HPLC. For HPLC, the column used for SFN detection was C18 (4.6 × 100 mm × 3 mm, Gemini−Nx plus C18, Phenomenex, USA) and the mobile phase used consisted of 0.2% of trifluoroacetic acid (TFA) pH 2 and acetonitrile (42:58, v/v) with the flow rate of 1.0 mL/min. The temperature of column and wavelength used for drug detection were 25 °C and 254 nm, respectively. The injected volume of the drug solution was 20 µL. Base-catalyzed method to cleave the amide bond was used to release the MTX. Briefly, 20 µg UnTHCPSi-PEI-MTX NPs were suspended in 1 mL of 0.1 M of NaOH solution at a reaction temperature of 70 °C for 8 h. Then the supernatant was obtained after centrifugation and the concentration of MTX was determined by HPLC. The column used for the MTX detection was C18 (4.6 × 100 mm × 3.5 mm, Zorbax C18, Agilent, USA) and the mobile phase used consisted of 0.2 M of Na2HPO4: 0.2M citric acid (2:1) and acetonitrile (90:10, v/v) with the flow rate of 1.0 mL/min. The temperature of column and wavelength used for drug detection were 25 °C and 302 nm, respectively. The injected volume of the drug solution was 20 µL. 28 ACS Paragon Plus Environment

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The payload of AS1411 was quantified by UV-Vis (UV-1600 PB Spectrophotometer, VWR). The concentration of the AS1411 solution was obtained by testing the UV absorbance (Abs) at 260 nm (OligoAnalyzer program; http://www.idtdna.com/Scitools (accessed December 22, 2015)). Briefly, the AS1411 solution was prepared in parallel and the Abs was tested and then converted into concentration of AS1411 (C1). Then, 20 µg SFN-loaded UnTHCPSi-PEI-MTX@AS1411 NPs were suspended in 1 mL (V) solution of AS1411 (Milli-Q water or HBSS buffer). Finally, the supernatant was obtained by centrifugation at 13,000g for 5 min, and the AS1411 concentration of obtained supernatant was C2. The equation for calculating the loading degree of AS1411 is as follow:

= AS1411

Payload-Depended

C1 − C2 ×V Mass of NPs

Surface

Charge

Inversion.

The UnTHCPSi-PEI-

MTX@AS1411 NPs was suspended in relevant solution (Milli-Q water or HBSS buffer) in parallel, with the concentration of 20 µg/mL and volume of 1 mL. Then, the AS1411 was added based on different mass ratios of the NPs. The obtained suspension was treated by ultrasonication and the ζ-potential was measured by Zetasizer Nano ZS. Cell Culturing. The MDA-MB-231 breast cancer cells and NIH 3T3 fibroblasts were cultured in cultured 75 cm2 culture with flasks (Corning Inc. Life Sciences, USA) in a standard BB 16 gas incubator (Heraeus Instruments GmbH, Germany) set at 95% humidity, 5% CO2, and 37°C. MDA-MB-231 cells were cultured in standard RPMI 1640 media and NIH 3T3 fibroblasts were cultured in DMEM, both supplemented with 10% (v/v) fetal bovine serum (FBS), 1% non-essential amino acids, 1% L-glutamine, penicillin (100 IU/mL), and streptomycin (100 mg/mL) (all from HyClone, USA). Cells’ subculturing was conducted at 29 ACS Paragon Plus Environment


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80% confluency, harvested prior to cell passaging and each experiment with trypsinPBS−ethylenediaminetetraacetic acid. In Vitro Cytotoxicity. To evaluate the biosafety of the NPs, the viability of the MDA-MB231 and NIH 3T3 fibroblast cells was assessed by measuring their ATP activity after exposure to the NPs. 100 µL of the cell suspensions in cell media at a concentration of 2 × 105 cells/mL were seeded in 96-well plates and allowed to attach overnight. After removal of the cell media, the wells were washed twice with HBSS−HEPES buffer (pH 7.4) and then 100 µL of the tested NPs at the relevant concentrations were added. After incubation, the reagent assay (100 µL; CellTiter-Glo Luminescent Cell Viability Assay, Promega, USA) was added to each well to assess the ATP activity. The luminescence was measured using a Varioskan Flash (Thermo Fisher Scientific Inc., USA). Positive (1% Triton X-100) and negative HBSS−HEPES buffer (pH 7.4) controls were also used and treated similarly as described above. At least three independent measurements were conducted for each experiment. Confocal Imaging and FACS. For confocal fluorescence microscopy imaging, MDAMB-231 and 3T3 fibroblast cells were seeded in 8-chamber slides (Nunc Lab-Tek II Chamber Slide System, Thermo scientifi c, Inc., USA). For cell seeding, 200 µL of the cells suspension (2.5 × 104 cells/mL) were added to each chamber. After incubation for 24 h, the cells were washed twice with HBSS–HEPES buffer (pH 7.4). 200 µL of FITC labeled-UnTHCPSi-PEIMTX@AS1411 NPs in HBSS–HEPES buffer (pH 7.4) with the concentration of 10 µg/mL were added to each chamber and then the samples were incubated for relevant time. After that, the cells were washed with HBSS–HEPES buffer to remove non-interacting NPs. The cell membrane was stained with CellMaskTM DeepRed (Life Technologies, USA) by 30 ACS Paragon Plus Environment

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incubating the cells at 37°C for 3 min. Then, the cells were washed twice with the HBSS– HEPES buffer and fixed with 2.5% glutaraldehyde at room temperature for 20 min. Finally, the glutaraldehyde was washed away and the cells were stored with 200 µL of HBSS–HEPES buffer (pH 7.4). The cells were observed under confocal fluorescence microscope (Leica inverted SP5 II HCS A) using Ar (488 nm), HeNe (590 nm) and HeNe (633 nm) lasers. The images were analyzed using ImageJ 1.47v (National Institute of health, USA). For the cell uptake flow cytometry analysis, MDA-MB-231 and 3T3 fibroblast cells were seeded in 6-well plates (Corning Inc. Life Sciences, USA). For cell seeding, 2.5 mL of the cells suspension (2 × 10 5 cells/mL) were added to each well. The cell culturing process was based on the aforementioned method. After that, 1.5 mL of FITC-labeled UnTHCPSi-PEIMTX@AS1411NPs in HBSS–HEPES buffer (pH 7.4) with the concentration of 10 µg/mL were added to each well and then the samples were incubated for relevant time. After removing the NPs suspensions and washing twice with HBSS–HEPES buffer, the cells were harvested and treated with trypan blue to quench the fluorescence of NPs adhered on the cell’s surface. Flow cytometry was performed with a LSR II flow cytometer (BD Biosciences, USA) with a laser excitation wavelength of 488 nm using a FACSDiva software. 10,000 events were obtained for each sample. Relevant data were analyzed and plotted using Flowjo software (Tree Star Inc., USA). At least three independent measurements were conducted for each experiment. The comparison of the NPs’ fluorescence intensity between different kinds of cell lines (A and B) is according to the following equation:



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Fluorescence intensity of NPs in A Fluorescence intensity of control of A ! " = Fluorescence intensity of NPs in B Fluorescence intensity of control of B

Receptor Competition Assay. For this study, after cell culturing as aforementioned, 1.5 mL of the relevant inhibitors (AS1411, FA and AS1411+FA) in HBSS−HEPES buffer (pH 7.4) with the concentration of 1 µg/mL were added to each well and then the samples were incubated for 15 min in 37°C. Then the buffer was removed and the samples were washed twice with HBSS−HEPES buffer (pH 7.4), and 1.5 mL of the mixture of FITC-labeled UnTHCPSi-PEI-MTX@AS1411 NPs (10 µg/mL) with relevant inhibitors (2 µg/mL) were added to each well and then the samples were incubated for 12 h in 37°C. The samples were harvested and treated with trypan blue to quench the fluorescence of the NPs adhered on the cell’s surface before cell uptake flow cytometry analysis. At least three independent measurements were conducted for each experiment. The inhibition ratio of X (InRX) was calculated as follows:

Inhibition Ratio X =

Mean FITC, AX − Mean FITC, A Control Mean FITC, AUnTHCPSi − PEI − MTX@AS1411 − Mean FITC, AControl

× 100%

Cell Growth Inhibition. The cell growth inhibition studies of the developed nanocomposites was monitored by measuring the antiproliferation effect of the free MTX, SFN, AS1411, the combination of MTX, SFN and AS1411, and drug-loaded UnTHCPSi-PEI-


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MTX@AS1411 NPs using the same method explained above for the cytotoxicity studies. At least three independent measurements were conducted for each experiment. Statistical Analysis. At least three independent experiments were performed to obtain the result as the mean ± standard deviation (SD). Statistical significance of the data was analyzed by a one-way analysis of variance (ANOVA) with Bonferroni post-test. The significance level was set at probabilities of *p < 0.05, **p < 0.01 and ***p < 0.001.

Acknowledgement. Prof. H. Zhang acknowledges Jane and Aatos Erkko Foundation (grant no. 4704010) for financial support. Prof. H.A. Santos acknowledges financial support from the Academy of Finland (decisions no. 252215 and 281300), the University of Helsinki Research Funds, the Biocentrum Helsinki, and the European Research Council under the European Union’s Seventh Framework Programme (FP/2007–2013, Grant No. 310892). The authors thank the Electron Microscopy Unit and the Flow Cytometry Unit of the Institute of Biotechnology, University of Helsinki, for providing the necessary laboratory facilities and assistance. The assistance and guidance from Dr. Dongfei Liu, Dr. Antti Rahikkala, Patrícia Figueiredo and Flavia Fontana are also acknowledged.

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