Enhancing Cellular Uptake and Doxorubicin Delivery of Mesoporous

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Enhancing cellular uptake and doxorubicin delivery of mesoporous silica nanoparticles via surface functionalization: effects of serum Shakiba Shahabi, Svea Döscher, Tobias Bollhorst, Laura Treccani, Michael Maas, Ralf Dringen, and Kurosch Rezwan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b09483 • Publication Date (Web): 12 Nov 2015 Downloaded from http://pubs.acs.org on November 15, 2015

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ACS Applied Materials & Interfaces

Enhancing cellular uptake and doxorubicin delivery nanoparticles via surface functionalization: effects of serum Shakiba Shahabi a, Svea Döscher a, Tobias Bollhorst a, Laura Treccani b a, c Dringen , Kurosch Rezwan

a b

of a*

mesoporous

silica

, Michael Maas a,c *, Ralf

Advanced Ceramics, University of Bremen, Am Biologischen Garten 2, 28359 Bremen, Germany Centre for Biomolecular Interactions Bremen and Centre for Environmental Research and

Sustainable Technology, Faculty 2 (Biology/Chemistry), University of Bremen, Leobener Strasse, NW2, 28359 Bremen, Germany c

MAPEX Center for Materials and Processes, University of Bremen, Germany

*Corresponding authors: Dr. Laura Treccani, Dr. Michael Maas Advanced Ceramics, University of Bremen, Am Biologischen Garten 2, 28359 Bremen, Germany

E-Mail: [email protected], [email protected] Tel: +49 421 218 64938 Fax: +49 421 218 64932

Abstract In this study, we demonstrate how functional groups on the surface of mesoporous silica nanoparticles (MSNPs) can influence the encapsulation and release of the anticancer drug doxorubicin, as well as cancer cell response in the absence or presence of serum proteins. To this end, we synthesized four differently functionalized MSNPs with amine, sulfonate, polyethylene glycol or polyethylene imine functional surface groups, as well as one type of antibody-conjugated MSNP for specific cellular targeting and characterized these MSNPs regarding their physicochemical properties, colloidal stability in physiological media and uptake and release of doxorubicin in vitro. Then, the MSNPs were investigated for their cytotoxic potential on cancer cells. Cationic MSNPs could not be loaded with doxorubicin and did therefore not show any cytotoxic and antiproliferative potential on osteosarcoma cells, although they were efficiently taken up into the cells in the presence or absence of serum. In contrast, substantial amounts of doxorubicin were loaded into negatively charged and unfunctionalized MSNPs. Especially sulfonate functionalized doxorubicin-loaded MSNPs were efficiently taken up into the cells in the presence of serum and showed an accelerated toxic and antiproliferative potential compared to unfunctionalized MSNPs, antibody-conjugated MSNPs and even free doxorubicin. These findings stress the high importance of the surface charge as well as of the protein corona for designing and applying nanoparticles for targeted drug delivery.

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Key words Doxorubicin, Drug delivery, Mesoporous silica nanoparticles, Osteosarcoma, Protein corona, Surface functionalization

1. Introduction For more than a decade, the surface chemistry of nanoparticles (NPs) has been considered a key factor directly affecting the interactions of NPs with cells. However, recent advances in understanding the behavior of NPs in biological media challenge the significance and predictability of this paradigm. It is well established that at physiological conditions, biological systems are generally not exposed to bare NPs. Instead, prior to cellular uptake, NPs become engulfed in a protein corona 1, 4

the complex mixture of distinct proteins in the biological media.

interactions.

which reflects

The protein corona defines the final

surface properties, aggregation rate and hydrodynamic size of NPs 7-8

1-3

1, 5-6

as well as their cellular

However, it is worth noting that although cells only come in contact with corona-

covered NPs, the final biological behavior might be indirectly influenced by the surface chemistry of NPs,

2, 9-10

5, 11

as it can have a significant impact on corona composition and evolution.

In our study,

we show how functional groups on the surface of mesoporous silica NP (MSNP) can be engineered and exploited to tune drug encapsulation rate, drug release and cell responses in the presence or absence of serum proteins. MSNPs have emerged as promising materials for drug, DNA or siRNA delivery 12-16 as a result of their unique properties, e.g., straightforward synthesis, biocompatibility, low degradation pathways in the biological milieu, optical transparency, water dispersibility, physiochemical stability, adjustable pore size, tunable pore morphologies, facile functionalization chemistry for bioconjugation and particularly 17-23

their high surface area and high pore volume.

For the detailed study of the effects of serum on the

performace of a model drug delivery system, we synthesized MSNP with different functional groups which were grafted to the surface of the particles using the sol-gel technique. Next to the unfunctionalized MSNP, we introduced amine (NH2), sulfonate (SO3H), polyethylene imine (PEI) or polyethylene glycol (PEG) groups to generate functionalized MSNPs (Scheme 1) with well-controlled stability and surface charge. Furthermore, MSNP were conjugated to the antibody CD11c for specific cellular targeting. The functionalized MSNPs were used for doxorubicin (Dox) loading, which was selected as a model payload due to its chemotherapeutical relevance, in particular for osteosarcoma bone tumor treatment.

24-25

The pH-responsive mechanism of Dox release can potentially be

strategically exploited to control payload release upon internalization of the particles into the lysosomal compartment of human osteosarcoma cells (MG-63).23, 26 In order to quantify NP uptake, experiments were also carried out with functionalized fluorescent mesoporous silica NP (FLMSNP, Scheme S1). Here, the dye rhodamine B isothiocyanate (RBITC) was covalently incorporated in the silica matrix during NP synthesis, without changing any relevant properties of the MSNPs like size and surface chemistry.


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Our experiments demonstrate that in the absence of serum, the chemical functionalization of MSNP surfaces has a significant effect on the cellular uptake of the particles and the cellular responses in accordance to established trends. In contrast, in the presence of serum proteins, only sulfonate functionalized MSNP were more efficient than the unfunctionalized MSNP, antibody-conjugated MSNP or even free Dox to cause cell toxicity and mediates antiproliferative effects on osteosarcoma cells.

Scheme 1. Schematic illustration and TEM micrograph of mesoporous silica nanoparticle (MSNP), its functionalization with amine, sulfonate, polyethylene imine (PEI) or polyethylene glycol (PEG) groups and their loading with doxorubicin (Dox). The highest possible Dox loading was obtained for sulfonate functionalized MSNP. Unfunctionalized MSNP, MSNP-PEG and MSNP-AB presented a good Dox loading, while for MSNP-NH2 and MSNP-PEI only a negligible Dox loading was quantified.



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2. Materials and Methods 2.1. Chemicals For particle synthesis, functionalization and characterization, tetraethyl orthosilicate (TEOS, >99%, lot no. BCBK1670V), an aqueous solution of cetyltrimethylammonium chloride (CTAC, 25 wt %in water, lot no. STBD9961V), diethanolamine (DEA, lot no. MKBR7116V), rhodamine B isothiocyanate (RBITC, mixed isomers, lot no. MKBJ9031V), 3-aminopropyl-triethoxysilane (APTES, >98%, lot no. BCBH2173V),

(3-thiolpropyl)

trimethoxy-silane

(MPS,

95%,

lot

no.

SHBD3265V),

4-(N-

maleimidomethyl) cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (sulfo-SMCC, lot no. 061M1263V), bovine serum albumin (BSA, 99% protein, molecular weight 66.5 kDa, lot no. SLBG1645V), and absolute ethanol (>98%, lot no. SZBB1570V) were purchased from Sigma Aldrich (Germany). Doxorubicin hydrochloride (Dox, code D2975000, Batch 6.1, VWR, Germany), 3-(trihydroxysilyl)-1propane sulfonic acid (HSPSA, 30-35% in H2O, lot no. 1246512, ABCR, Germany), Pierce® BCA protein

assay

(lot

no.

OC

185009,

Thermo

Scientific,

Germany),

2-

[Methoxy(polyethyleneoxy)propyl]trimethoxy-silane (PEG-silian, 90%, 6-9 PE-units, ABCR, Germany) and trimethoxysilylpropyl-polyethyleneimine (TSPEI, 50% in isopropanol, GELEST, USA) were obtained from different suppliers as specified. Data on the exact molecular weight of PEI chains in the used commercial TSPEI are unfortunately not available. More information on the characterization of this PEI-silane from the same company are presented by Dacarro et al.27 For specific cell targeting anti-mouse CD11c antibody (lot no. E029528 and E03537-1630) as wells as anti-mouse CD11c FITCconjugated (lot no. E00153-1631) were purchased from eBioscience, Germany. Cell culture tests were carried out on human osteosarcoma cells (MG-63, obtained at passage 98, lot no. 2006399, ATCC, Germany). Dulbecco/Vogt modified Eagle’s minimal essential medium (DMEM, high glucose, lot no. 1654723), antibiotic–antimycotic solution (lot no. 1209917), Alexa Fluor®488 ®

phalloidin (AF488, 2U/ml, lot no. 1151587) and LysoTracker (DND-22, lot no. 791512) were from Invitrogen (Germany). Fetal calf serum (FCS, lot no. 010M3395), paraformaldehyde (PFA, lot no. 53260), phosphate buffered saline (PBS, lot no. SLBF5741V), TritonTM X-100 (lot no. MKBL5839V), sodium chloride (NaCl, lot no. 038K00451), sodium pyruvate (lot no. SLBH3762V), 4',6-diamidino-2phenylindole (DAPI, 0.5 µg/ml, lot no. 1242642), chlorpromazine hydrochloride (lot no. 027M3145), wortmannin (lot no. 023M4072V), nystatin (lot no. 020M13491) and dimethyl sulfoxide (DMSO, lot no. 055K01033) were supplied by Sigma-Aldrich (Germany). The cell proliferation assay WST-1 (lot no. 14310400, Roche Diagnostics, Germany) and nicotinaminde adenine dinucleotide (NADH, lot no. 4L015586, Applichem, US) were purchased from the suppliers as specified. Double deionized water (ddH2O) with a conductivity 515 nm) for AF488 and FS15 (λex: 535-558 nm, λem: > 590 nm) for RBITC and Dox. Particle uptake and distribution within the cells were analysed using fluorescent FLMSNPs. As Dox showed an intense red fluorescence, it was possible to study the cellular uptake of Dox loaded MSNPs as well as of free Dox using fluorescence microscopy. As for each type of FLMSNP or Dox-loaded MSNP the results obtained after incubations for 2, 4 or 6 h were almost similar, only fluorescence microscopy images of the cells after 0.5 or 6 h exposure to the FLMSNPs or Dox-loaded MSNPs are presented and the images after 2 or 4 h are not shown. Cellular localization of particles or Dox within lysosomes was investigated by co-incubation with the ®

®

fluorescent dye LysoTracker . To avoid possible autofluorescence of Thermanox coverslips in the lysosomal co-localization experiments, MG-63 cells were cultured on glass coverslips (lot. no. 0983, VWR, Germany) and incubated with FLMSNPs, Dox loaded MSNPs or free Dox for 0.5, 2, 4 or 6 h. ®

Afterwards cells were incubated with LysoTracker (75 nM) for 1 h, then washed twice with PBS and fixed with PFA. Cell actin cytoskeleton was stained with AF488 as previously described.41 The applied ®

LysoTracker is a blue fluorescent dye that stains the acidic lysosomes. Hence, cellular co-localization of the FLMSNPs, Dox loaded MSNPs or free Dox (all with red fluorescence) with LysoTracker® will yield a magenta overlap in the merged images. Due to similarities in the results for the different incubation periods investigated, only representative fluorescence microscopy images obtained after 6 h incubations of the MG-63 cells with FLMSNPs in media with or without serum are presented.

2.5.4. Quantification of cellular particle uptake To estimate cellular association of FLMSNPs, after each sampling point the MG-63 cells were trypsinized and washed with PBS (three times with a centrifugation each time at 600 g for 5 min). Subsequently, the cell pellet was resuspended in PBS and analysed by fluorimetry in black well plates (Greiner Bio-One, Germany) at the excitation wavelength of 544 nm and the emission wavelength of 590 nm using a microplate reader (Chameleon, HIDEX, Turku, Finland). Background fluorescence (cells without NPs) was substracted and the obtained fluorescence intensity units were normalized to the fluorescence intensity of the applied FLMSNPs dispersions.

2.5.5. Inhibition of endocytosis In order to identify possible endocytosis pathways for MSNPs uptake into MG-63 cells, the cellular fluorescence was quantified after incubation of the cells for 2 h with FLMSNPs in the presence or absence of the endocytosis inhibitors, chlorpromazine (40 µM), wortmannin (600 nM) or nystatin (10

µM). In addition, cells were incubated at 4 °C with MSNPs for 2 h to study the possible temperaturedependent inhibition of cellular FLMSNPs accumulation. Subsequently the cellular content of particles was analyzed by fluorimetry of cell pellets as described above. The data are given as a percentage of the respective FLMSNPs-treated cells at 37 °C, with DMSO and without inhibitors (control).



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2.5.6. Determination of nuclear Dox internalization To quantify the nuclear Dox fluorescence intensity, the Carl Zeiss Zen software was used to analyse the fluorescence microscopy images of the cells captured at the end of each sampling point (0.5, 2, 4 or 6 h). The signal intensity of the red channel (reflecting Dox internalization in 20 separate nuclei) was determined after application of Dox loaded MSNPs or free Dox to the cells.

2.6. Statistical analysis Quantitative data (zeta potential, drugs L.E. and release, cellular particle uptake, nuclear Dox fluorescence as well as the results assessed by BCA, WST-1 and LDH assays) are given as mean ± standard deviation of values obtained in three independently performed experiments. The statistical analysis was performed using the software Minitab 16 (Minitab Inc., Pennsylvania). The data was subjected to one-way analysis of variance (ANOVA) followed by Dunnett’s method for multiple comparisons. p-values below 0.05 (p

Enhancing Cellular Uptake and Doxorubicin Delivery of Mesoporous

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