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Chemical Modifications of Porous Carbon Nanospheres Obtained from Ubiquitous Precursors for Targeted Drug Delivery and Live Cell Imaging Sutanu Kapri, Rahul Majee, and Sayan Bhattacharyya ACS Sustainable Chem. Eng., Just Accepted Manuscript • Publication Date (Web): 04 Jun 2018 Downloaded from http://pubs.acs.org on June 4, 2018
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ACS Sustainable Chemistry & Engineering
Chemical Modifications of Porous Carbon Nanospheres Obtained from Ubiquitous Precursors for Targeted Drug Delivery and Live Cell Imaging Sutanu Kapri, Rahul Majee, and Sayan Bhattacharyya* Department of Chemical Sciences, and Centre for Advanced Functional Materials, Indian Institute of Science Education and Research (IISER) Kolkata, Mohanpur - 741246, India *Email for Correspondence:
[email protected] Abstract Cost-effective anti-cancer delivery vehicles that can ensure controlled and targeted transportation of drug molecules are pertinent to modern day biomedical applications. Minimally toxic 9-13 nm diameter porous carbon nanospheres (PNs) were synthesized by oxidative cutting of porous carbon matrices (PCs) obtained by carbonization of pasture grass, human hair and sucrose. Among them, the grass-derived PNs (PN-G) with superior surface area, porosity and graphitic content demonstrates a significant loading of the drug both by chemical binding and physisorption. Polyethylenimine (PEI) and folic acid (FA) functionalization maintain therapeutic efficacy of the drug doxorubicin (DOX) to the targeted folate receptor (FR) over-expressed human cervical cancer cells (HeLa) and human breast cancer cells (MDA-MB-231) through receptor mediated endocytosis while FR deficient normal cells (human embryonic kidney 293) exhibits substantially lower endocytosis under identical conditions. Moreover, upon loading cellimpermeable propidium iodide (PI), the PNs display superior activity towards near infrared (NIR) live cell imaging in HeLa cells whereby due to a higher binding affinity of PI with the nucleic acids, the PI-to-PN energy transfer quenched fluorescence is recovered. This dual functionality of controlled and targeted drug delivery and photobleaching resistant live cell imaging by the cost-effective PNs has larger implications in nanomedicine research and technology. Keywords: Porous carbon nanosphere; Drug delivery; Live cell imaging; Folic acid; Polyethylenimine
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Introduction Two of the several challenges in nanomedicine,1 are the cell-specific receptor mediated targeted and controlled delivery of drug inside the cells, and fluorescence imaging of live cells at a wavelength range where the biomolecules do not absorb and emit light at similar wavelengths as the fluorophores. An intelligent theranostic anti-cancer nanosystem avoids the pitfalls of chemotherapy for clinical translation,2 whereby the drug/dye molecules are encapsulated inside the nanocarrier to prevent premature release into the cellular microenvironment,3 and reduces the toxicity towards healthy cells.4 A required size 10-100 nm of spherical nanoparticles (NPs) allows favorable entry into the intracellular compartments by endocytosis,5 whereas drug/dye encapsulation can be facilitated by their chemical conjugation and/or physical adsorption onto the NPs. Cell-specific targeting can be achieved by a surface-attached ligand such as folic acid, biotin or antibody,2,6 and controlled drug release can be triggered by an internal or external stimuli such as pH.3,4 In recent years, plenty of nanocarriers such as polymeric NPs, mesoporous silica and carbon were demonstrated to improve prolong blood circulation time, drug solubility, enhanced therapeutic efficacy and eventually reduced side effects.7-9 Each of the aforementioned nanoparticulate systems has their own advantages and disadvantages. For example, mesoporous silica NPs with tunable porosity are known for intracellular drug release,10,11 but prime issues related to their clinical success involve complicated synthesis steps, cytotoxicity and leakage of drug from the nanocarrier.12 From these perspectives, mesoporous carbon materials are emerging candidates for controlled and targeted intracellular drug/dye delivery and biomedical imaging probes, owing to their unique properties such as high biocompatibility, surface area to volume ratio and graphitic nature for drug/dye adsorption.4,8,13,14 On the other hand, carbon materials can be very useful in assisting fluorescence imaging of live cells,15 wherein the autofluorescence and light scattering of biomolecules can be circumvented. While fluorescence imaging is one of the most powerful techniques for both in vitro and in vivo monitoring of cellular functions,16,17 most of the fluorescent markers face limitations owing to their absorption at 300-500 nm and emission at 400-550 nm wavelengths where ultraviolet excitation damages the cells due to lengthy irradiation.18 Interference from the cellular matrix further leads to limited tissue penetration, low signal-to-noise ratios and reduced photostability.17,19,20 Thus it is a pressing need to use fluorescent markers that absorb at 2 ACS Paragon Plus Environment
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wavelengths >500 nm and emit in the NIR region, 650–900 nm. A majority of the cell permeable far-red or NIR emitting commercial organic dyes are overpriced (Supporting Information, Table S1) which limits the imaging procedures from being economical. Also, several known hydrophilic dyes are impermeable to the live cells.18 In this context, PI is a cost-effective cell impermeant fluorescent dye with λexcitation = 538 nm and λemission = 617 nm; interacts efficiently with the nucleic acids. Especially, PI can detect the dead cell population by intercalating between the DNA base pair and widely used in several biological experiments and assays of DNA content in cell cycle analysis, apoptosis assay and flow cytometry experiments to evaluate cell viability.21 Like several organic dye molecules, PI can penetrate the cell wall only if conjugated within the nanocarriers, whereby the tailored carbon materials play an important role.4,22 Except carbon dots and graphene oxide, most of the carbon nanomaterials such as singlewalled carbon nanotubes, graphene and porous carbon tend to agglomerate in physiological conditions because of their relative hydrophobic nature, hence difficult to be taken up by the cells which in turn minimize their therapeutic efficacy.23-27 Although carbon nanomaterials are known to possess excellent biocompatibility even after prolonged exposure in vivo,28,29 the easy bioclearance of these nanomaterials is inhibited by their chemical inertness inside the body, especially for irregular shapes and sizes >8 nm. The macrophages present in vivo can easily engulf these NPs which are excreted from the body through liver and spleen instead of them undergoing biodegradation. Also a majority of mesoporous carbon NP based drug delivery vehicles suffer from conventional preparation costs, micron-sized particles, irregular shapes and low scalability.30-32. To address these challenges, uniform ~12 nm diameter porous nanospheres (PNs) of carbon with good water dispersibility, biocompatibility, semi-graphitic nature, high surface area and uniform pore diameter were synthesized from porous carbon (PC) obtained by chemical oxidation of low cost omnipresent precursors such as pasture grass, human hair and sucrose. The micron-sized PCs were converted to PNs by an oxidative scalable process using piranha (3:1, H2SO4:H2O2) at room temperature which also introduces oxygen containing functional groups to generate water dispersibility. Our method has advantages over other chemical oxidation processes such as Hofmann,33 Hummers,34 or Tour’s,35 methods in which there is an abundance of water soluble metal ions, the release of greenhouse gases and hazardous hydrocarbons in thermal exfoliation of graphitic materials,36 or the industrially expensive oxygen gas assisted piranha oxidation of graphite.37 Among the PNs, the one derived from pasture grass 3 ACS Paragon Plus Environment
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(PN-G) is capable of better encapsulation of DOX and PI on the surface and inside the pores via π-π interaction and hydrogen bonding, thus allowing the transport and release of these molecules for drug delivery and live cell staining. The pH responsive temporal release profile is triggered by endosomal pH. The targeted release in the folate receptor (FR) over-expressed human cervical cancer cells (HeLa) and human breast cancer cells (MDA-MB-231) is achieved by designing a complex of DOX loaded PNs with polyethylenimine (PEI) and folic acid (FA). The costeffective PNs with DOX/PI loading show exceptional ability in mitigating the normal (human embryonic kidney (HEK-293) cells during cancer cell treatment and enables live cell imaging without interference from the biomolecules, respectively. Results and discussion Structural Characterization The pasture grass, human hair, sucrose and cetyltrimethyl ammonium bromide (CTAB) assisted sucrose derived PCs are abbreviated as PC-G, PC-H, PC-S and PC-S-CTAB, respectively (see Table 1 for abbreviations). The corresponding PNs are PN-G, PN-H, PN-S and PN-S-CTAB, and the DOX loaded PN-G named as PN-G-DOX, PN-G-DOX/PEI and PN-GDOX/PEI-FA, respectively. The PI loaded PNs are named as PN-G-PI, PN-H-PI, PN-S-PI and PN-S-CTAB-PI, respectively. The PCs obtained by calcination of the carbonized products from dried bio-waste precursors consist of micron sized porous matrix (Figure S1). The PNs obtained by piranha oxidation of the PCs and subsequent purification in phosphate buffered saline (PBS) buffer solution are spherical 10-25 nm diameter carbon nanospheres (Figure 1). Although the field emission scanning electron microscope (FESEM) images show clustered particles, the diameter of the PNs are distinctly visible from the atomic force microscope (AFM) images with corresponding height profiles. The diameter range of PN-G, PN-H and PN-S-CTAB is 10-13, 912 and 9-10 nm, respectively. In the absence of surfactant, PN-S shows lesser monodispersity with an average cluster diameter of 25 nm (Figure 1g). Figure 1i shows that the representative transmission electron microscope (TEM) image of synthesized PN-G nanocarrier with average diameter of ~11 nm and inset showing the corresponding size distribution profile. The high resolution TEM (HR-TEM) image shows their spherical and amorphous morphology (Figure 1j) although the selected area electron diffraction pattern shows diffraction spots corresponding to (002) plane of hexagonal graphite (Figure 1k).4,33 Due to the presence of N-containing groups in
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human hair, pasture grass, and CTAB, a modest quantity of nitrogen remains in the PNs (Table S2). The graphitic domains in the PNs are suitable for the loading of DOX/PI molecules, since most of the aromatic drug molecules interact by π-π staking with the basal plane of graphene layers. To investigate the degree of graphitization, Raman spectra were recorded for PNs and PCs, shown in Figure 2a and Figure S2, respectively. The D band at 1337-1340 cm-1 is due to the breathing mode of κ-point phonons with A1g symmetry related to the structural defects created by the attachment of hydroxyl and epoxide groups on the carbon basal plane. The G band at 1598 cm-1 is due to the in plane vibrations of sp2 hybridized carbon atoms and a doubly degenerate phonon mode (E2g symmetry) at the Brillouin zone center.38,39 The ratio of the intensities of the D and G bands (ID/IG) of PC-G, PC-H, PC-S and PC-S-CTAB are 0.97, 1.09, 1.07 and 1.13, respectively. Oxidation decreases the sp2 domains, whereby ID/IG increases to 0.99, 1.52, 1.30 and 1.36 for PN-G, PN-H, PN-S and PN-S-CTAB, respectively. In addition, oxidative cutting introduces the functional groups on PN as envisaged by fourier transform infrared (FTIR) spectra (Figure 2b) which show the appearance of vibrational bands at 3438, 1630 and 1091 cm-1 corresponding to –O-H stretching, aromatic –C=C stretching and C-OH stretching, respectively.34,40 The process introduces epoxy, hydroxyl and carboxylic acid groups both on the walls and inside the pores of PNs.41 Prior to oxidation, all PCs except PC-S-CTAB show O-H stretching vibration of lower intensity at 3430 cm-1 and C-O stretching at 1083 cm-1 (Figure S3). The bands at 2961 and 2860 cm-1 are due to C-H stretching vibration of methylene group, and those at 1613, 1213 and 1034 cm-1 are attributed to C=C stretching and C-O stretching vibrations, respectively.42 After DOX adsorption onto PN-G to form PN-G-DOX, the intense peaks at 1715, 1610, 1410 and 1071 cm-1 are ascribed to carbonyl group (quinine and ketone) vibrational modes in DOX skeleton (Figure 2c).43 When PEI is grafted, the broadened peaks at 3500-3150 cm−1 and 1635-1450 cm−1 are assigned to –N-H and –NH2 vibration modes, respectively, indicating a successful grafting. The covalent attachment of FA with PN-GDOX/PEI to form PN-G-DOX/PEI-FA results in a broadened peak at 1630 cm-1 due to the amide bond between –NH2 functional group of PEI and carboxylic group of FA, as well as the appearance of characteristic bands of FA at 1500-500 cm-1.34,44 Since DOX is preserved within the pores of PN-G-DOX, PN-G-DOX/PEI and PN-G-DOX/PEI- FA nanocarriers, the 13 keto group of the DOX network consistently shows the band at 1715 cm-1. The stepwise appearance 5 ACS Paragon Plus Environment
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of new vibrational bands suggests the successful encapsulation of DOX and attachment of FA. During oxidative cutting, HSO4- and atomic oxygen are generated from the piranha solution. While HSO4- intercalates into the graphitic layer, atomic oxygen reacts with the edge site of the graphitic layer of PCs to produce smaller entities (Figure 2d). The N2 sorption isotherms of PCs and PNs along with the corresponding BJH pore size distributions are shown in Figure S4 and Figure 3, respectively. The surface area, pore volume, and average pore diameter are listed in Table 1. According to IUPAC classification the PCs display a typical type IV adsorption isotherm with H4 type hysteresis loop.45 PNs display similar isotherm characteristics albeit with lower surface area and pore volume. The N2 sorption curves (Figure 3) have a steep capillary condensation step at a relative pressure of P/P0 0.45–0.9 which indicates capillary condensation of N2 inside the mesopores with a narrow pore distribution of 3.6-3.8 nm.46,47 After DOX and PI loading, PN-G-DOX/PEI-FA, PN-G-PI and PN-G-DOX show a decrease in surface area suggesting the engross of foreign molecules by the mesoporous scaffolds (Figure 3c and Figure S4c) X-ray diffraction (XRD) patterns of the representative PCG, PN-G and PN-G-DOX/PEI show two well-resolved diffraction peak at 2θ ≈ 24o and 43.5o corresponding to (002) and (101) crystallographic planes of hexagonal graphite, respectively (Figure 4a).31 After oxidative cutting of PC-G and PEI grafting on PN-G-DOX, the (002) reflection is slightly shifted to lower angle indicating the introduction of oxygen functional groups within the graphitic planes.48 In vitro Drug Loading and Release Studies Among all the nanocarriers, PN-G loads the highest quantity of any drug (Figure S5) owing to its better porosity, pore volume and excellent dispersibility in physiological media (Figure S6) PN-G-DOX/PEI was obtained by coating PEI on DOX adsorbed PN-G. It is well established that the cellular toxicity of PEI with high MW (~25 kDa) is mainly attributed to their proton sponge effect in contrast to low MW (
