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Enhanced Intracellular Delivery and Tissue Retention of Nanoparticles by Mussel-Inspired Surface Chemistry Kai Chen, Xiaoqiu Xu, Jia Wei Guo, Xuelin Zhang, Songling Han, Ruibing Wang, Xiaohui Li, and Jianxiang Zhang Biomacromolecules, Just Accepted Manuscript • Publication Date (Web): 29 Sep 2015 Downloaded from http://pubs.acs.org on October 4, 2015
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Enhanced Intracellular Delivery and Tissue Retention of Nanoparticles by Mussel-Inspired Surface Chemistry Kai Chen1,‡, Xiaoqiu Xu1,2,‡, Jiawei Guo1, Xuelin Zhang1, Songling Han1, Ruibing Wang3, Xiaohui Li2, Jianxiang Zhang1,* 1
Department of Pharmaceutics, College of Pharmacy, Third Military Medical University, Chongqing 400038,
China 2
Institute of Materia Medica, College of Pharmacy, Third Military Medical University, Chongqing 400038,
China 3
State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences,
University of Macau, Taipa, Macau, China
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KEYWORDS: biomimetic • catechol • drug delivery • intracellular uptake • nanoparticle • tissue retention ABSTRACT: Nanomaterials have been broadly studied for intracellular delivery of diverse compounds for diagnosis or therapy. Currently it remains challenging for discovering new biomolecules that can prominently enhance cellular internalization and tissue retention of nanoparticles (NPs). Herein we report for the first time that a mussel-inspired engineering approach may notably promote cellular uptake and tissue retention of NPs. In this strategy, the catechol moiety is covalently anchored onto biodegradable NPs. Thus fabricated NPs can be more effectively internalized by sensitive and multidrug resistance tumor cells as well as some normal cells, resulting in remarkably potentiated in vitro activity when an antitumor drug is packaged. Moreover, the newly engineered NPs afford increased tissue retention post local or oral delivery. This biomimetic approach is promising for creating functional nanomaterials for drug delivery, vaccination, and cell therapy.
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INTRODUCTION Intracellular delivery of various therapeutics and contrast agents is crucial for prevention, diagnosis, and treatment of most diseases. Nanomaterials have been extensively utilized to deliver a plethora of bioactive compounds into cells, which include small molecular drugs, peptides/proteins, and nucleic acids.1-5 So far, nanoparticles (NPs) with different biophysicochemical properties, such as varied size, shape, composition, surface charge and chemistry, and biological functions, have been studied for drug delivery, gene therapy, biosensing, and molecular imaging.6-11 Biologically functionalized NPs can also be used to modulate stem cells or regulate immune cells for cell-based therapies or tissue regeneration.12,
13
To fully realize these diverse
applications, NPs must reach their intracellular destination, which necessitate the capability to traverse the biological barrier of the plasma membrane. Generally, cellular uptake of NPs is mediated by membraneembedded receptors or by interacting with the lipid bilayer via hydrophobic and/or electrostatic interactions.14 Accordingly, different biochemical strategies have been developed for rational surface modifications or coatings to increase cell binding affinities, which are frequently implemented by physical coating or covalent conjugation of cell penetrating peptides, receptor ligands, antibodies, and aptamers.1,
4, 15, 16
Although some
progress has been achieved based on these approaches, their in vivo applications and clinical translation remains challenging, mainly due to synthetic cost, in vitro and in vivo stability, loss or attenuation of efficiency in the complex biological milieu, regulatory hurdles, or potential immunogenicity after repeated dosing.17-20 Further, exploring new molecules that can target specific endocytic pathways is highly necessary in certain cases such as nonviral gene delivery and vaccination. As a result, there is still unmet demand for discovering novel biomolecules that can effectively enhance cellular internalization of NPs for both in vitro and in vivo utilization. 3,4-Dihydroxyphenylalanine is a critical component of adhesive proteins secreted by marine mussels, and its catecholic functionality plays an important role in the adhesion of mussels to wet surfaces in the ocean.21, 22 Materials containing the catechol moiety afford adhesive, coating, and anchoring properties, which can bind strongly to various inorganic and organic surfaces.22-29 Recent studies also reveal that mussel-mimetic hydrogels with dopamine (DOPA, also has the catechol group) units display superior adhesive forces towards the 3 ACS Paragon Plus Environment
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epididymal fat pad, the external liver surfaces, the inside surface of blood vessels, and atherosclerotic plaques.30, 31
These results demonstrated that structured materials decorated with DOPA moieties may have additionally
increased absorption, binding, and adhesion capabilities to different surfaces and tissues. This was largely realized by combined effects of oxidation-dependent conjugation and hydrogen-binding (H-bonding). Notably, oxidation of catechol in DOPA may produce the quinone structure that further forms cross-linking via aryl-aryl coupling.32 Alternatively, quinones may react with amine-containing biomolecules through Michael-type addition reactions.33 Also, DOPA and quinones could form H-bonding with polysaccharide moieties and/or amino acid residues of the cell membrane. On the basis of these issues, herein we hypothesize that surface engineering of NPs with DOPA may enhance their intracellular uptake, taking advantages of the superior adhesive and binding capability of the catechol moiety (Figure 1A). As a proof of concept, DOPA was covalently conjugated onto NPs via a hydrophilic linker of polyethylene glycol (PEG). Thus decorated NPs showed significantly enhanced internalization by different cell lines, which in turn led to remarkably potentiated efficacy of paclitaxel (PTX) payload against both sensitive and multidrug resistance (MDR) tumor cells. EXPERIMENTAL SECTION Materials. β-Cyclodextrin (β-CD) and lecithin (from soybean) were purchased from Tokyo Chemical Industry Co., Ltd (Tokyo, Japan). 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000]
(DSPE-PEG-COOH)
and
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-
[methoxy(polyethylene glycol)-2000] (DSPE-PEG) were purchased from Avanti Polar Lipids, Inc. (USA). 2Methoxypropene (MP), dopamine hydrochloride (DOPA·HCl), and nocodazole were obtained from Sigma (St. Louis,
USA).
N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide
hydrochloride
(EDC·HCl)
and
N-
hydroxysuccinimide (NHS) were purchased from Fluka (USA). Paclitaxel (PTX) was supplied by Xi’an Haoxuan Biological Technology Co., Ltd (Xi’an, China). Pyridinium p-toluene sulfonate (PTS) was obtained from Acro Organics. Poly(lactide-co-glycolide) (PLGA, 50:50) with intrinsic viscosity of 0.50-0.65 was purchased from Polysciences, Inc (USA). Penicillin, streptomycin, fetal bovine serum (FBS), and Dulbecco’s Modified Eagle’s Medium (DMEM) were purchased from HyClone (Waltham, MA, USA). RPMI1640 medium 4 ACS Paragon Plus Environment
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was obtained from Gibco (USA). Cy5 NHS ester and Cy7.5 NHS ester were obtained from Lumiprobe, LLC. (USA). 4’,6-Diamidino-2-phenylindole (DAPI) and LysoTracker® Red were purchased from Invitrogen (USA). 1,1’-Dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate (Dil) was purchased from Beyotime (China). Cytochalasin D was supplied by Cayman Chemical Company (USA). Synthesis of DOPA-Conjugated DSPE-PEG. DOPA-conjugated DSPE-PEG (DSPE-PEG-DOPA) was synthesized by coupling reaction. To this end, 100 mg of DSPE-PEG-COOH (35 µmol) was dissolved into 40 mL PBS (pH 7.4). Then, 20 mg of NHS (176 µmol) and 67.3 mg EDC·HCl (350 µmol) were added. After 12 h of activation at room temperature, 66.4 mg of DOPA·HCl (350 µmol) was added into the reaction mixture, followed by magnetic stirring for 24 h under the protection of nitrogen at 4ºC. The obtained polymer was purified by dialysis for 24 h using dialysis tubing (molecular weight cut-off of 1000 Da) in deionized water and then collected by freeze-drying. Synthesis of Acetalated β-CD. Acetonation of β-CD was performed in the presence of excess amount of MP, using PTS as a catalyst.34 Briefly, 20 mL of MP (210 mmol) was added into 100 mL of anhydrous DMSO containing 5 g β-CD (4.4 mmol), into which 80 mg of PTS was added. After 3 h of acetalation under magnetic stirring at room temperature, the reaction was terminated by adding 2 mL of triethylamine into the mixture. The acetalated product (Ac-bCD) was precipitated from water, collected by filtration, thoroughly washed with deionized water, and lyophilized to a white powder. Preparation of Various Nanoparticles. A modified nanoprecipitation/self-assembly method was employed to prepare Ac-bCD NPs.35 Briefly, 50 mg of Ac-bCD was dissolved in 2 mL acetonitrile. Lecithin and DSPEPEG at the molar ratio of 7:3 were dispersed in 0.8 mL ethanol, and then 19.2 mL deionized water was added. Thus obtained aqueous dispersion was heated to 65ºC for 1 h. Then, the Ac-bCD solution was added into the preheated aqueous solution dropwise (1 mL/min) under gentle stirring, followed by vortexing for 3 min. After 2 h of incubation, the mixture was cooled to room temperature. The solidified NPs were collected by centrifugation at 16, 000 rpm for 10 min, rinsed with deionized water three times. Following similar procedures, Ac-bCD or PLGA NPs based on DSPE-PEG and DSPE-PEG-DOPA at different molar ratios were fabricated. 5 ACS Paragon Plus Environment
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Similarly, PTX-loaded and Cy5 or Cy7.5-labeled NPs were produced. The content of PTX in NPs was quantified by high performance liquid chromatography (HPLC, LC-20A, Shimadzu), while Cy5 and Cy7.5 were quantified by UV-Vis spectroscopy. Measurements. 1H NMR spectra were recorded on a Bruker BioSpin-600 spectrometer operating at 600 MHz. FT-IR spectra were acquired on a Perkin-Elmer FT-IR spectrometer (100S). Dynamic light scattering and zeta-potential measurement of NPs in aqueous solution was performed with a Malvern Zetasizer Nano ZS instrument at 25ºC. Transmission electron microscopy (TEM) observation was carried out on a TECNAI-10 microscope (Philips, Netherland) operating at an acceleration voltage of 80 kV. In Vitro Hydrolysis Study. In vitro hydrolysis of Ac-bCD NPs at 0% or 60% DOPA was conducted at 37°C in PBS with pH 5 or pH 7.4. Briefly, about 0.5 mg of freshly prepared NPs was dispersed in PBS. After incubation for different periods of time, the transmittance at 500 nm was measured. In Vitro Release Tests. In vitro release experiments were performed in PBS at pH 5 or pH 7.4. Briefly, 0.1 mL of aqueous solution containing 0.25 mg freshly prepared Ac-bCD NPs loaded with PTX was placed into dialysis tubing, which was immersed into 30 mL of PBS and incubated at 37°C. At predetermined time points, 5.0 mL of release medium was withdrawn, and the same volume of fresh PBS was replenished. The PTX concentration was quantified by HPLC. Cell Culture. B16F10, HepG2, MCF-7, MDA-MB-231, MOVAS, and RAW264.7 cells were cultured in 96-well plates at a density of 1×104 cells per well in 100 µL growth medium containing 10% (v/v) FBS, 100 U/mL of penicillin, and 100 µg/mL of streptomycin. All cells were incubated at 37°C in a humidified atmosphere of 5% CO2 for 24 h before the addition of various NPs. B16F10, HepG2, MDA-MB-231, MOVAS, and RAW264.7 cells were cultured in RPMI 1640 medium, while MCF-7 cells were cultured in EME medium. Intracellular Uptake Study by Fluorescence Microscopy. B16F10 and MDA-MB-231 cells were separately seeded in a 35 mm dish with 20 mm cover glasses at a density of 2×105 cells per well in 2 mL growth medium. Cells were incubated at 37ºC with 5% CO2 for 24 h. Then the culture medium was replaced by 2 mL of fresh medium containing Cy5-labeled NPs and incubated for 2, 4, 8, and 12 h, respectively. Before 6 ACS Paragon Plus Environment
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observation, cells were stained with LysoTracker Red (50 nM) for 1.5 h and Dil (20 µM) for 15 min. After washing with PBS, cells were counterstained with DAPI. Confocal laser scanning microscopy (CLSM) observation was performed on a fluorescence microscope (LSM780NLO, Zeiss, Germany). Quantification of Cellular Uptake by Flow Cytometry. Uptake efficiency of different cell lines for NPs was quantified by the fluorescence activated cell sorting (FACS) technique. Specifically, B16F10, MDA-MB231, RAW264.7, and MOVAS cells were planted in 6-well plates with 5×105 cells per well in 2 mL of DMEM containing 10% FBS (v/v) 24 h before the experiments. Generally, the treated cells were washed three times with PBS (pH 7.4), followed by trypsinization, centrifugation (1000 rpm for 5 min), and washing with PBS (pH 7.4), and then re-suspended in 200 µL of FACS solution. The cell uptake of Cy5-labeled NPs was quantified by flow cytometry (FACSVerse, Becton Dickinson, USA). Approximate 10, 000 events were acquired per sample, and the data were analyzed using FlowJo. Forward and side light scatter gates were normally set to exclude dead cells, debris, and cell aggregates. In each study, three independent experiments were performed. Effects of Various Treatments on Cellular Uptake. B16F10 cells were planted in 6-well plates with 5×105 cells per well in 2 mL of DMEM containing 10% FBS (v/v) 24 h before the uptake experiment. The cells were pre-incubated at 4ºC for 30 min or pre-treated with various inhibitors (nocodazole at 10 µg/mL; cytochalasin D at 5 µg/mL) for 30 min, followed by the addition of Cy5-labeled NPs. After 4 h of incubation, the cells were washed three times with PBS. Then, the cells were trypsinized, centrifuged, re-suspended in 200 µL of FACS solution, and analyzed by flow cytometry (FACSVerse, Becton Dickinson, USA). Three separate experiments were performed. Cytotoxicity Evaluation. Cells were incubated at 37ºC in a humidified atmosphere of 5% CO2 overnight, and then they were treated with the medium containing various concentrations of NPs with or without DOPA coating for 24 h. Cell viability was quantified by the MTT assay, and the experiment was repeated three times. Values of the half maximal inhibitory concentration (IC50) were calculated by curve fitting using Originpro 7.0. In Vitro Antitumor Activity of PTX-Loaded Nanoparticles. All cells were incubated under standard conditions for 24 h. Cells were then treated with the medium containing various PTX formulations (including 7 ACS Paragon Plus Environment
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free PTX and PTX-loaded Ac-bCD/DSPE-PEG NPs with or without DOPA coating) at different doses of PTX. After 24 h of incubation, the cell viability was quantified by the MTT method, and the IC50 values were calculated based on three separate experiments. Study on the Intratumor Retention of Nanoparticles. All animal care and experimental protocols were performed in compliance with the Animal Management Rule of the Ministry of Health of the People’s Republic of China (No. 55, 2001) and the guidelines for the Care and Use of Laboratory Animals of the Third Military Medical University (Chongqing, China). Four-week-old male BALB/c athymic nude mice (16-20 g) were acclimatized for 1 week before experimentation. To establish MCF-7 human breast cancer xenografts, a suspension of MCF-7 cells at a density of 9×106 cells/mouse was subcutaneously injected in the right flank of nude mice. When tumors reached an average volume of 100-150 mm3, tumor bearing mice were randomly assigned into 3 groups (n = 3). Then, intratumor injection of 50 µL of aqueous solution containing Cy7.5-labeled NPs (10 mg/mL) was implemented. Real-time fluorescence imaging was performed and the fluorescence intensity at various time points was determined by a living imaging system (IVIS Spectrum, PerkinElmer, USA). After 72 h, the mice were sacrificed, both tumors and main organs were resected for further analysis. Retention of DOPA-Coated NPs in the Intestine. After one week of acclimatization, male C57BL/6 mice (4 weeks old, 16-20 g) were randomly assigned into three groups (n = 3). Post 12 h of fasting, 300 µL of PBS (0.01 M, pH 7.4), PBS containg Cy7.5-labeled PLGA NP at 0% DOPA (4 mg/mL) or PBS containing Cy7.5/PLGA NP at 60% DOPA (4 mg/mL) was separately administered by gastric gavage. At 12 and 24 h post administration, mice were sacrificed and differnt tissues were resected for ex vivo fluorescence imaging by a living imaging system (IVIS Spectrum, PerkinElmer, USA). Statistical Analysis. Statistical analysis was performed by SPSS15.0. The one-sample KolmogorovSmirnov test was used to determine whether samples were in normal distribution. If samples were in normal distribution, the one-way ANOVA test was applied for multiple comparison of statistical analysis. If samples were not in normal distribution, then the Wilcoxon rank-sum test was performed. In the one-way ANOVA test, 8 ACS Paragon Plus Environment
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if data were homoscedastic, the LSD test was employed, while the Dunnett T3 test was used if data were heteroscedastic. The p < 0.05 is considered to be statistical significance. RESULTS AND DISCUSSION Fabrication and Characterization of Nanoparticles. As a proof of concept, biodegradable NPs with peripheral PEG chains were employed in this study, since this hydrophilic and biocompatible surface chemistry is frequently utilized to provide colloidal stability and prolonged blood circulation for different nanomaterials. Nevertheless, PEG coating by either noncovalent or covalent approaches can also impede intracellular uptake and subsequent trafficking, and therefore innovative strategies are imperative for both drug and gene delivery. DSPE-PEG was used to fabricate NPs with PEG coating. As a hydrophobic group, DSPE may facilitate hydrophobic interactions of DSPE-PEG with the lipophilic core composed of Ac-bCD, thus forming a hydrophilic monolayer. DOPA-conjugated DSPE-PEG (DSPE-PEG-DOPA, the molecular weight of PEG is 2 kDa) was firstly synthesized by coupling reaction between carboxyl-terminated DSPE-PEG (DSPE-PEGCOOH) and excessive DOPA, using EDC and NHS as catalysts (Figure 1B). The product was purified by dialysis against deionized water, and then lyophilized. Characterization by Fourier transform infrared and 1H NMR spectroscopy showed disappearance of free carboxyl and appearance of catechol in the obtained material (Figure S1), indicating successful conjugation. Calculation by the ratio of proton signals at 6.7-7.1 ppm (due to aryl protons of DOPA) to those at 3.4-3.6 ppm (corresponding to ethylene in PEG) revealed a conjugation efficiency of 85%. On the other hand, acetalated β-CD (Ac-bCD) was used as a model hydrophobic carrier, since previous studies have demonstrated that this type of materials may serve as safe and effective pHresponsive carriers for intracellular delivery of various therapeutics in different cell lines.36-41 Ac-bCD was synthesized by kinetically controlled acetalation of β-CD in the presence of excessive amount of 2methoxypropene.34 According to the 1H NMR spectrum (Figure S1B), the molar ratio of linear acetal to cyclic acetal was 1.65 for the synthesized Ac-bCD. Both control and DOPA-coated NPs were fabricated through a modified nanoprecipitation/self-assembly method that is a well-established approach for processing hydrophobic materials into NPs of different sizes.35 It 9 ACS Paragon Plus Environment
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should be noted that for simplicity, the molar percentage of DSPE-PEG-DOPA was calculated according to the content of PEG. For NPs without DOPA coating, PEG was considered as 100%. Independent of the content of DSPE-PEG-DOPA introduced, well-defined spherical NPs could be successfully prepared, as illustrated by typical TEM images of Ac-bCD/DSPE-PEG NPs with varied contents of DSPE-PEG-DOPA (abbreviated as the molar percentage of DOPA, Figure 1C). Consistent with previous results,42 staining by phosphotungstic acid showed core-shell structure for the obtained NPs (the last image in Figure 1C), implying that DSPEPEG/DSPE-PEG-DOPA was peripherally anchored around the hydrophobic core of Ac-bCD NPs. Measurement by dynamic light scattering indicated that NPs with mean size of 189 to 210 nm were manufactured, when the content of DSPE-PEG-DOPA varied from 0% to 80% (Figure 1D and Figure S2). As evidenced by the determined zeta-potential values, surface charge of DOPA-coated NPs was slightly decreased as compared to the control NPs without DOPA (Figure 1D). Consequently, the incorporation of DOPA had no significant influence on physicochemical properties of resulting NPs. Intracellular Uptake of DOPA-Coated NPs in Different Cell Lines. Then cellular uptake profiles of DOPA-coated Ac-bCD NPs were observed by CLSM and quantified by FACS. Using Cy5-labeled NPs, we firstly examined their internalization in B16F10 melanoma cells. After incubation for various periods of time, both the time and DOPA content related cellular uptake behaviors could be clearly observed (Figure 2A and Figure S3). For the control NPs without DOPA coating, weak green fluorescence appeared in cells at 2 h, which was gradually intensified with prolonged incubation. By contrast, remarkably strong fluorescence was observed when DOPA-decorated NPs at the same dose of Cy5 were used, particularly at 4 and 8 h. Furthermore, difference in the fluorescence intensity could be found when the content of coated DOPA was varied. Irrespective of the incubation time, the maximal intensity was observed at 60% DOPA. Based on these intuitive results, cellular uptake of various NPs by B16F10 cells was further quantified by FACS via flow cytometry. After treatment with different NPs for various times, we could observe that uptake efficiency was changed with incubation time and DOPA coating (Figure 2B). Quantitative analysis of repeated results indicated that internalization of NPs was gradually increased when NPs were coated with DOPA varying 10 ACS Paragon Plus Environment
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from 0%, 20%, 40%, 60%, to 80% (Figure 2C). Nevertheless, whereas initial increase in DOPA was beneficial for cellular uptake, impairing effect was observed when the DOPA content was above 60%. Consistent with CLSM observation, the optimal internalization efficiency was achieved at 60% DOPA. In this case, 3.3-, 4.0-, and 7.2-fold increase in mean fluorescence intensity was reached post 2, 4, and 8 h of treatment with DOPAdecorated NPs, respectively, as compared to that of control NPs without DOPA. When the dose of Ac-bCD NPs was increased from 10 to 20 and 40 µg/mL, almost the similar uptake profiles were examined by flow cytometry (Figure 2D). Accordingly, this DOPA-facilitated cellular uptake performance was largely independent of the NPs dosage. In other words, the maximal uptake efficiency was achieved at 60% DOPA for all examined doses. We also found similar uptake profiles of different NPs in a MDR human breast cancer cell line of MDAMB-231, for which both CLSM observation and FACS quantification showed the superior performance at 60% DOPA (Figure 3A-B and Figure S4). Besides tumor cells, DOPA was able to promote cellular internalization in some normal cells. As exemplified by mouse vascular smooth muscle (MOVAS) cells, at 60% DOPA, the internalized NPs were considerably increased in a time dependent manner (Figure S5A). We also found enhanced internalization of Ac-bCD NP with 60% DOPA in RAW264.7 murine macrophage cells (Figure S5B). For different cell lines, it was notable that the exact uptake kinetics was different, which should be attributed to their distinct phagocytosis activities. According to previous studies, DOPA-containing materials adhere to organic substrates via H-bonding with polar components and covalent coupling via reactive quinone moieties.22, 23, 31
In addition, it has been found that cellular uptake of peptides, polymers, and NPs may be facilitated by H-
bonding between their amino/hydroxyl groups and either sugar or protein units present in cell membrane.43-45 In view of the presence of amide and phenolic hydroxyl groups in DOPA-coated NPs, the combined effects of Hbonding and oxidation-dependent conjugation might account for the enhanced internalization in various cells observed herein. Also, CLSM observation uncovered a time-dependent subcellular trafficking of both DOPA-coated and control NPs. In B16F10 cells, green fluorescence was largely distributed peripherally at 2 h (Figure 2A). 11 ACS Paragon Plus Environment
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Staining of late endosomes/lysosomes with LysoTracker Red showed partly co-localized red and green fluorescence (yellow regions, Figure 2E), indicating endocytosis at an early stage. After 4 h of treatment, however, intensive fluorescence near the nucleus could be observed. The colocalization of green and red fluorescence, as indicated by significant yellow areas, revealed a considerable number of NPs were transported via endosomes/lysosomes (Figure 2E). This is especially true in the case of DOPA free NPs. At 8 h, disseminated green fluorescence throughout the whole cytoplasm implied endolysosomal escape of some NPs. Nevertheless, bright fluorescence appeared near the membrane after 12 h of treatment. This might be partially related to exocytosis through egress of NPs from late endosomes/lysosomes, as delineated by the presence of yellow fluorescence, which was also documented for both smooth muscle cells and tumor cells previously;46, 47 while other processes responsible for this remains elusive. Likewise, the time-dependent, endolysosomal transport was found in MDA-MB-231 cells (Figure 3A, C). As compared to NPs without DOPA, however, the relatively distinct accumulation of green fluorescence for NPs with 60% DOPA suggested that other cytoplasmic transportation pathways might be involved for DOPA-anchored NPs. We then interrogated biological processes that dominate internalization of these NPs in B16F10 cells, by perturbating the endocytosis-mediated uptake under various conditions, in combination with quantification by FACS (Figure 2F). Firstly, energy depletion was implemented by pretreatment at 4°C. Regardless of NPs with different DOPA contents, dramatically reduced uptake efficiency could be found. This coincides with the previous findings on various cells that endocytic uptake is energy-dependent.48 Intracellular uptake was also remarkably attenuated when cells were pretreated with cytochalasin D, a potent inhibitor that can suppress caveolae uptake,48 suggesting the caveolae route was also concerned in endocytosis of NPs by B16F10 cells. Likewise, decreased uptake efficiency was detected post treatment with nocodazole that can inhibit microtubule dynamic instability. This effect might be related to impaired cytoplasmic trafficking along microtubules, because the decreased cytosolic transport may lead to increased exocytosis.5 Since energy depletion and transport inhibitors did not completely inhibit cellular uptake of NPs, multiple pathways such as caveolaeindependent endocytosis, macropinocytosis, as well as other non-identified endocytic routes may be involved in 12 ACS Paragon Plus Environment
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the internalization of NPs by B16F10 cells. It is worth noting that DOPA-coated NPs were more sensitive to all the applied perturbations, particularly uptake and transport inhibitors, when NPs with 60% DOPA were concerned. Consequently, at least to a certain degree, DOPA-coated NPs might be internalized and transported in different manners as compared to DOPA-free NPs, and this is in line with CLSM observation. Of note, coating of DOPA had no remarkable influence on the cytotoxicity of Ac-bCD NPs. After incubation with B16F10 cells for 6 h, Ac-bCD NPs with DOPA varying from 20%, 40%, 60%, to 80% displayed comparable cell viability at 500 µg/mL (Figure 4A). Besides, similar dose-dependent changes in viability of B16F10 cells were detected for Ac-bCD NPs at 0% and 80% DOPA, after 24 h of treatment (Figure 4B). There was no significant different between the values of IC50 for Ac-bCD NPs at 0% and 80% DOPA (Figure 4C). Also, we found high cell viability for both RAW264.7 and MDA-MB-231 cells (Figure S6A-B), and the corresponding IC50 was as high as 614.1 and 635.0 µg/mL (Figure S6C), respectively. Additionally, this implied that DOPA-coated Ac-bCD NPs did not activate macrophages,49 in line with the fact that no acidic byproducts are produced upon hydrolysis of NPs based on acetalated polysaccharides.34, 36, 39, 50 In Vitro Anticancer Activities of PTX-Containing Nanoparticles. Unambiguously, above results demonstrated enhanced intracellular uptake of NPs by peripherally decorating with DOPA. Accordingly, conjugation of DOPA at the terminal of PEG chains on NPs is promising to conquer the PEG dilemma by simultaneously providing colloidal stability and maintaining effective internalization. To examine whether the enhanced internalization may contribute to therapeutic delivery capability, in vitro experiments were performed using PTX as a model drug. PTX-loaded Ac-bCD NPs with or without DOPA coating were also fabricated by the modified nanoprecipitation/self-assembly method, resulting in PTX nanomedicines with comparative size, shape, and surface charge (Figure 5A-C). The PTX loading content was 9.8% and 9.7% for NPs at 0% and 60% DOPA, respectively. Of note, similar pH-responsive hydrolysis and in vitro PTX release profiles were observed for Ac-bCD NPs at 0% and 60% DOPA (Figure S7), indicating that surface coating of DOPA had no significant effects on pH-sensitivity of resulting NPs. This may be attributed to the fact that for this type of core-shell NPs, their sensitivity is mainly determined by the core material. 13 ACS Paragon Plus Environment
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Initially, in vitro antitumor activity of PTX/Ac-bCD NPs was evaluated in sensitive tumor cell lines of B16F10 and human hepatocarcinoma cells (HepG2). After 24 h of incubation with various doses of PTXcontaining NPs, cell viability was determined by MTT assay. Clearly, the inhibition capability was enhanced with increase in PTX doses for all formulations (Figure 5D). The pharmacological activity of PTX was significantly increased by packaging into Ac-bCD/DSPE-PEG NPs, consistent with our previous finding that emulsion-based PTX nanomedicines from acetalated cyclodextrins were able to potentiate the payload efficacy.39, 40 At 60% DOPA, further improved PTX efficacy could be observed, as compared to that without DOPA (Figure 5D). Likewise, we found additionally enhanced antitumor activity against two MDR human breast cancer cells, i.e. MCF-7 and MDA-MB-231, when PTX was encased into NPs with 60% DOPA, in comparison to that of DOPA-free NPs (Figure 5E). The IC50 was calculated based on the data of dose-dependent cell viability (Table 1). For B16F10 cells, the IC50 value was 27.0, 14.2, and 8.2 µg/mL for free PTX, PTX/Ac-bCD NPs at 0% DOPA, and PTX/Ac-bCD NPs at 60% DOPA, respectively; while it corresponded to 5.5, 3.2, and 2.2 µg/mL in the case of HepG2 cells. Both NPs were pharmacologically superior over free PTX, and there was significant difference between the nanomedicines at 0% and 60% DOPA. More prominent differences could be found for MDR cells. Whereas treatment by PTX/Ac-bCD NPs at 0% DOPA resulted in 3.3-fold decrease in IC50 against MCF-7 cells in comparison to free PTX, PTX/Ac-bCD NPs at 60% DOPA yielded 9.5 times of reduction. As for MDA-MB231 cells, The PTX nanomedicine at 0% and 60% DOPA afforded 5.6- and 9.4-fold decrease in the IC50 value, respectively, when compared with free PTX. Since DOPA coating did not affect pH-sensitivity of NPs as well as release profiles of PTX, the enhanced PTX activity should be largely contributed by surface DOPA. These results strongly suggested that surface coating of nanomedicines with DOPA is beneficial for their in vitro activity, primarily resulting from the enhanced intracellular delivery. Taken together, above results clearly demonstrated that surface decoration of NPs with well-determined contents of DOPA may remarkably enhance their cellular uptake, which in turn can potentiate the efficacy of their therapeutic payload. Besides, this type of nanosystems are promising for in vitro modulation, stimulation, 14 ACS Paragon Plus Environment
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or activation of different cells (such as stem/progenitor cells and immune cells) for cell therapy. Whereas this surface chemistry lacks specificity, this disadvantage can be partly overcome by tailoring the density of DOPA or combining with other targeting units. Enhanced Tumor Retention of DOPA-Coated Nanoparticles. Intrigued by these promising findings, we then interrogated whether surface coating of DOPA may enhance tissue retention of NPs by the strengthened adhesion capability, which is beneficial for local drug delivery, vaccination, or tissue repair via topical treatment. First, a tumor tissue was employed as a diseased model. For this purpose, the same dose of Cy7.5labeled Ac-bCD NPs with or without DOPA coating was separately injected into tumors in nude mice with MCF-7 xenografts. Real-time imaging at predetermined time points was performed, and representative images are illustrated in Figure 6A. It should be noted that the relative weak intensity immediately after injection was due to fluorescence quenching. For NPs without DOPA, the fluorescence was mainly located around the tumor at 24 and 48 h (particularly at 48 h), which indicated the diffusion of NPs out of the tumor in this case, owning to the high interstitial fluid pressure. By contrast, fluorescence was homogeneously distributed in the tumor injected with NPs with 60% DOPA. Moreover, relatively high intensity could be detected for NPs with 60% DOPA, as compared with those without DOPA, and there was significant difference at 48 h (Figure 6B). At both 24 and 48 h, the percentage of fluorescence at tumor sites of Ac-bCD NPs at 60% DOPA was significantly higher than that at 0% DOPA (Figure S8A). In line with this result, ex vivo imaging and quantification of the resected tumor tissues revealed that the fluorescence intensity at 60% DOPA was prominently higher than that at 0% DOPA (Figure 6C-D). The fluorescence intensity ratio of tumor to other main organs was 1.0 and 2.9 for Cy7.5/Ac-bCD NPs at 0% and 60% DOPA, respectively (Figure S8B). Additionally, NPs with 60% DOPA led to relatively low distribution in the examined major organs, especially in heart, liver, and spleen. These results substantiated that coating of NPs with DOPA may remarkably increase their intratumor retention after local injection, while simultaneously decrease undesirable distribution in other organs. This is particularly advantageous to enhance efficacy yet decrease side effects for local drug delivery or vaccination. 15 ACS Paragon Plus Environment
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Subsequently, to investigate whether DOPA coating may facilitate adhesion and retention in other tissues, we examined tissue distribution of Cy7.5-labeled NPs with or without DOPA after oral administration. As illustrated by ex vivo imaging (Figure 6E), only weak fluorescence could be observed in the intestine at 12 h after oral delivery in the case of Cy7.5 NP at 0% DOPA. This is consistent with the fact that orally administered microparticles will be largely eliminated from the gastrointestinal tract within 12-24 h. By contrast, dramatically stronger fluorescence was found in the intestine of mice treated with Cy7.5 NP at 60% DOPA. Quantitative analysis indicated significant difference in the intestinal fluorescence intensity between 0% DOPA and 60% DOPA groups (Figure 6F). Likewise, both intuitive and quantitative results suggested that fluorescence in the intestine of the 60% DOPA group was much more notable at 24 h, as compared to that of Cy7.5 NP at 0% DOPA (Figure 6G-H). On the other hand, fluorescence in other organs such as heart, liver, spleen, lung, and kidney was slightly increased at 60% DOPA, while there was no significant difference as compared to those at 0% DOPA. Since distribution in these organs are generally related to translocation of NPs by transcellular and paracellular pathways, these preliminary results indicated that DOPA coating did not affect absorption of NPs in the intestine. Nevertheless, these findings demonstrated that surface coating of NPs with DOPA may considerably enhance their retention in the intestine tissue. This function is especially advantageous to treat intestinal diseases (such as inflammatory bowel disease and colon cancer) or to achieve long-term mucosal immune response for oral vaccination. The enhanced tumor and intestinal retention of NPs by DOPA coating should be attributed to increased adhesion and anchoring on the extracellular matrix and augmented intracellular uptake in local cells, although the details require further exploration. CONCLUSIONS In summary, for the first time we demonstrated herein that biomimetic coating of NPs with DOPA is beneficial for enhancing their intracellular delivery, potentiating cellular activity against both sensitive and MDR tumor cells, as well as increasing retention in tumor and intestinal tissues. Nanomaterials engineered by this strategy may find applications in intracellular delivery of various therapeutics, local drug delivery, vaccination, and NPs-mediated cell therapy. 16 ACS Paragon Plus Environment
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A
B Zeta-potential (mV) Size (nm)
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250
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C D Figure 1. Engineering of high affinity nanoparticles (NPs). A, Schematic showing enhanced intracellular uptake of DOPA-coated NPs based on acetalated β-CD (Ac-bCD). EDC, N-(3-dimethylaminopropyl)-N′ethylcarbodiimide; NHS, N-hydroxysuccinimide. B, Synthesis of DOPA-conjugated DSPE-PEG (DSPE-PEGDOPA). C-D, TEM images (C) as well as average size and zeta-potential (D) of Ac-bCD NPs containing various contents of peripheral DOPA. Data are mean ± SE (n = 3).
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Control 0% DOPA 20% DOPA 60% DOPA 80% DOPA *** *** *** 6
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Control 20% DOPA
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0% DOPA 60% DOPA
100 80 60 40 20 0
4°C alasin D odazole ch Noc Cyto
E F Figure 2. Cellular uptake of Ac-bCD NPs peripherally coated with DOPA in murine B16F10 melanoma cells. A, Internalization of different NPs by B16F10 tumor cells after incubation for various periods of time. The cell membrane was stained by Dil, while the nucleus was counterstained with DAPI. B-C, Flow cytometric profiles (B) and quantitative results (C) showing time-dependent uptake of DOPA-coated Cy5/Ac-bCD NPs in B16F10 cells at 10 µg/mL of NPs. D, Quantification of uptake of DOPA-coated NPs by B16F10 at 20 µg/mL (the left panel) and 40 µg/mL (the right panel). E, Intracellular trafficking of NPs in B16F10 cells. The lysosome was stained with LysoTracker Red, while the nucleus was counterstained with DAPI. Scale bars, 20 µm. F, Effects of various treatments on internalization in B16F10 tumor cells. All data are mean ± SE (n = 3). For results in (C-D), the data at 0% DOPA were normalized to 1 at each time point. *p
