Article pubs.acs.org/Biomac
Synthesis and Cellular Uptake of Folic Acid-Conjugated Cellulose Nanocrystals for Cancer Targeting Shuping Dong,†,⊥ Hyung Joon Cho,‡,⊥ Yong Woo Lee,‡,§ and Maren Roman*,†,∥ †
Macromolecules and Interfaces Institute, ‡School of Biomedical Engineering and Sciences, §Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, and ∥Department of Sustainable Biomaterials, Virginia Tech, Blacksburg, Virginia 24061, United States S Supporting Information *
ABSTRACT: Elongated nanoparticles have recently been shown to have distinct advantages over spherical ones in targeted drug delivery applications. In addition to their oblong geometry, their lack of cytotoxicity and numerous surface hydroxyl groups make cellulose nanocrystals (CNCs) promising drug delivery vectors. Herein we report the synthesis of folic acid-conjugated CNCs for the targeted delivery of chemotherapeutic agents to folate receptor-positive cancer cells. Folate receptor-mediated cellular binding/uptake of the conjugate was demonstrated on human (DBTRG-05MG, H4) and rat (C6) brain tumor cells. Folate receptor expression of the cells was verified by immunofluorescence staining. Cellular binding/uptake of the conjugate by DBTRG-05MG, H4, and C6 cells was 1452, 975, and 46 times higher, respectively, than that of nontargeted CNCs. The uptake mechanism was determined by preincubation of the cells with the uptake inhibitors chlorpromazine or genistein. DBTRG-05MG and C6 cells internalized the conjugate primarily via caveolae-mediated endocytosis, whereas H4 cells internalized the conjugate primarily via clathrin-mediated endocytosis.
1. INTRODUCTION The selective delivery of chemotherapeutic agents to cancer cells by targeted nanoparticles has the potential to reduce or even eliminate the often severe side effects of these agents, including decrease in blood cell counts, mouth ulcers, hair loss, kidney damage, heart damage, nausea, and vomiting. In recent years, particle geometry has been recognized as an important factor in the effectiveness of nanoscale drug carriers. Elongated or filamentous nanoparticles have been shown to have distinct advantages over spherical ones in terms of surface area-tovolume ratio, rate of clearance from the body, and elimination mechanism.1−3 Cellulose nanocrystals (CNCs) are elongated glucose-based nanoparticles with mean aspect ratios of 10−100 depending on the cellulose source and the experimental conditions used in their preparation.4 In addition to their oblong geometry, the surface chemistry of CNCs, which is governed by cellulose’s numerous hydroxyl groups (3 per repeat unit), makes CNCs promising drug delivery vectors. The hydroxyl groups enable easy coupling of imaging, targeting, and therapeutic agents to the CNC surface and render the particle surface hydrophilic, which has been associated with delayed clearance from the blood stream by the mononuclear phagocyte system.5−10 Previously, we have shown that CNCs are nontoxic to human brain endothelial cells and that they exhibit nearly no nonspecific cellular uptake.11 In a more recent report, we have demonstrated their lack of cytotoxicity and nonspecific cellular uptake for a variety of other mammalian cells, including KB cells, mouse brain endothelial cells (bEnd.3), mouse macrophages (RAW 264.7), © 2014 American Chemical Society
benign and malignant human breast cancer cells (MCF-10A, MDA-MB-231, MDA-MB-468), human prostate cancer cells (PC-3), and rat brain tumor cells (C6).12 Using our synthesis method for fluorescently labeled CNCs,13 Mahmoud et al. showed that at physiological pH rhodamine B isothiocyanateconjugated CNCs, contrary to fluorescein isothiocyanate (FITC)-conjugated CNCs, are internalized by human embryonic kidney 293 cells (HEK 293) and Spodoptera f rugiperda cells (Sf9) and that they exhibit no cytotoxicity toward these two cell lines.14 For drug delivery applications, however, uptake of CNCs via specific cellular uptake pathways, such as receptor-mediated endocytosis, is preferable over nonspecific uptake because it enables the selective treatment of pathological cells. To this end, we report here for the first time the synthesis of folic acidconjugated CNCs and demonstrate in vitro their folate receptormediated uptake by human (DBTRG-05MG, H4) and rat (C6) brain tumor cells. Despite advances in treatment modalities and drug discovery, brain cancer is still frequently fatal. In a separate study, published elsewhere,15 we have shown that CNCs potentiate the destruction of cancer cells by irreversible electroporation (IRE), a promising new treatment modality for the nonthermal ablation of brain tumors.16−18 Specifically, we demonstrated that treatment of KB and MDA-MB-468 cells with folate receptor-targeted CNCs prior to application of a pulsed electric field causes a significant increase in the cytotoxic effect of Received: October 28, 2013 Revised: March 11, 2014 Published: March 26, 2014 1560
dx.doi.org/10.1021/bm401593n | Biomacromolecules 2014, 15, 1560−1567
Biomacromolecules
Article
suspension was sonicated under ice-bath cooling for 10 min at 40% output. Finally, the suspension of CNC-NH2 was centrifuged for 10 min at 4550g and the sediment was discarded. The concentration of the obtained aqueous suspension of CNC-NH2 was determined by gravimetric analysis and raised for the subsequent reaction by rotary evaporation. FITC Labeling of CNC-NH2. FITC labeling of CNC-NH2 was carried out in a buffer solution consisting of 240 mL deionized water, 41.1 g sucrose, 0.76 g EGTA, 3.52 g NaCl, and 7.64 g of Na2B4O7· 10 H2O. To this solution, 2 g of FITC was added under stirring and the flask was covered with aluminum foil to exclude light. Next, 50 g of an aqueous suspension of CNC-NH2 of 2 wt % was added under ice-bath cooling. After 4 h, 140 mL of buffer solution was added to dissolve excess FITC that had deposited on the flask wall. The reaction was allowed to proceed overnight in the dark. Then, the reaction mixture was transferred to dialysis tubing and dialyzed against deionized water. When diffusion of excess FITC from the reaction mixture had ceased according to visual inspection of the dialysis water, the suspension was sonicated under ice-bath cooling for 10 min at 40% output and centrifuged for 12 min at 4550g to remove any aggregates. The supernatant was again dialyzed against deionized water until the dialysis water did no longer show FITC UV−vis absorption peaks (∼2 days). The concentration of the obtained aqueous suspension of FITC-CNC, determined by gravimetric analysis, was generally close to 0.1 wt %. Folic Acid Conjugation of FITC-CNCs. Folic acid (0.12 g), EDC (0.054 g), and Sulfo-NHS (0.061 g) were added under stirring to 20 mL PBS. The pH of the reaction mixture was determined with a pH meter (Mettler Toledo S47 SevenMulti pH/conductivity meter with an Inlab 413 pH electrode) and adjusted to 7 by addition of 0.02 N NaOH. Then, 150 g of an aqueous suspension of FITC-CNC of about 0.1 wt % was added and the pH of the reaction mixture adjusted again to 7. The reaction was allowed to proceed for 40 h at room temperature under stirring in the dark. The reaction mixture was processed as described above by dialysis, sonication, centrifugation, and second dialysis until the dialysis water did no longer show folic acid UV−vis absorption peaks (∼2 days). FTIR Spectroscopy. FTIR absorption spectra were recorded with a Thermo Scientific Nicolet 8700 FTIR spectrometer from KBr pellets, prepared as follows, with a resolution of 4 cm−1 and 64 scans per sample. Small amounts of unreacted and reacted CNCs were isolated by freezedrying of aliquots of the respective aqueous suspensions. For KBr pellet preparation, 2 mg of sample was thoroughly ground in a mortar together with 98 mg of anhydrous KBr. The obtained powder was dried in a vacuum oven at 45 °C for several hours and then pressed into a pellet between two stainless steel bolts inserted from opposite ends into a stainless steel nut. FTIR spectra were recorded from the pellets after further drying in a vacuum oven at 45 °C for several hours. UV−vis Spectroscopy. UV−vis absorption spectra were recorded with a Thermo Scientific Evolution 300 UV−vis spectrometer from dilute solutions/suspensions of known concentration between 0.001 and 0.01 wt % in standard cuvettes with an optical path lengths of 1 cm. Atomic Force Microscopy (AFM). AFM images were recorded in ac mode in air with an Asylum Research MFP-3D-Bio atomic force microscope using standard Olympus silicon probes (OMCL-AC160TS) with a nominal tip radius and spring constant of
Synthesis and Cellular Uptake of Folic Acid-Conjugated Cellulose Nanocrystals for Cancer Targeting Shuping Dong,†,⊥ Hyung Joon Cho,‡,⊥ Yong Woo Lee,‡,§ and Maren Roman*,†,∥ †
Macromolecules and Interfaces Institute, ‡School of Biomedical Engineering and Sciences, §Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, and ∥Department of Sustainable Biomaterials, Virginia Tech, Blacksburg, Virginia 24061, United States S Supporting Information *
ABSTRACT: Elongated nanoparticles have recently been shown to have distinct advantages over spherical ones in targeted drug delivery applications. In addition to their oblong geometry, their lack of cytotoxicity and numerous surface hydroxyl groups make cellulose nanocrystals (CNCs) promising drug delivery vectors. Herein we report the synthesis of folic acid-conjugated CNCs for the targeted delivery of chemotherapeutic agents to folate receptor-positive cancer cells. Folate receptor-mediated cellular binding/uptake of the conjugate was demonstrated on human (DBTRG-05MG, H4) and rat (C6) brain tumor cells. Folate receptor expression of the cells was verified by immunofluorescence staining. Cellular binding/uptake of the conjugate by DBTRG-05MG, H4, and C6 cells was 1452, 975, and 46 times higher, respectively, than that of nontargeted CNCs. The uptake mechanism was determined by preincubation of the cells with the uptake inhibitors chlorpromazine or genistein. DBTRG-05MG and C6 cells internalized the conjugate primarily via caveolae-mediated endocytosis, whereas H4 cells internalized the conjugate primarily via clathrin-mediated endocytosis.
1. INTRODUCTION The selective delivery of chemotherapeutic agents to cancer cells by targeted nanoparticles has the potential to reduce or even eliminate the often severe side effects of these agents, including decrease in blood cell counts, mouth ulcers, hair loss, kidney damage, heart damage, nausea, and vomiting. In recent years, particle geometry has been recognized as an important factor in the effectiveness of nanoscale drug carriers. Elongated or filamentous nanoparticles have been shown to have distinct advantages over spherical ones in terms of surface area-tovolume ratio, rate of clearance from the body, and elimination mechanism.1−3 Cellulose nanocrystals (CNCs) are elongated glucose-based nanoparticles with mean aspect ratios of 10−100 depending on the cellulose source and the experimental conditions used in their preparation.4 In addition to their oblong geometry, the surface chemistry of CNCs, which is governed by cellulose’s numerous hydroxyl groups (3 per repeat unit), makes CNCs promising drug delivery vectors. The hydroxyl groups enable easy coupling of imaging, targeting, and therapeutic agents to the CNC surface and render the particle surface hydrophilic, which has been associated with delayed clearance from the blood stream by the mononuclear phagocyte system.5−10 Previously, we have shown that CNCs are nontoxic to human brain endothelial cells and that they exhibit nearly no nonspecific cellular uptake.11 In a more recent report, we have demonstrated their lack of cytotoxicity and nonspecific cellular uptake for a variety of other mammalian cells, including KB cells, mouse brain endothelial cells (bEnd.3), mouse macrophages (RAW 264.7), © 2014 American Chemical Society
benign and malignant human breast cancer cells (MCF-10A, MDA-MB-231, MDA-MB-468), human prostate cancer cells (PC-3), and rat brain tumor cells (C6).12 Using our synthesis method for fluorescently labeled CNCs,13 Mahmoud et al. showed that at physiological pH rhodamine B isothiocyanateconjugated CNCs, contrary to fluorescein isothiocyanate (FITC)-conjugated CNCs, are internalized by human embryonic kidney 293 cells (HEK 293) and Spodoptera f rugiperda cells (Sf9) and that they exhibit no cytotoxicity toward these two cell lines.14 For drug delivery applications, however, uptake of CNCs via specific cellular uptake pathways, such as receptor-mediated endocytosis, is preferable over nonspecific uptake because it enables the selective treatment of pathological cells. To this end, we report here for the first time the synthesis of folic acidconjugated CNCs and demonstrate in vitro their folate receptormediated uptake by human (DBTRG-05MG, H4) and rat (C6) brain tumor cells. Despite advances in treatment modalities and drug discovery, brain cancer is still frequently fatal. In a separate study, published elsewhere,15 we have shown that CNCs potentiate the destruction of cancer cells by irreversible electroporation (IRE), a promising new treatment modality for the nonthermal ablation of brain tumors.16−18 Specifically, we demonstrated that treatment of KB and MDA-MB-468 cells with folate receptor-targeted CNCs prior to application of a pulsed electric field causes a significant increase in the cytotoxic effect of Received: October 28, 2013 Revised: March 11, 2014 Published: March 26, 2014 1560
dx.doi.org/10.1021/bm401593n | Biomacromolecules 2014, 15, 1560−1567
Biomacromolecules
Article
suspension was sonicated under ice-bath cooling for 10 min at 40% output. Finally, the suspension of CNC-NH2 was centrifuged for 10 min at 4550g and the sediment was discarded. The concentration of the obtained aqueous suspension of CNC-NH2 was determined by gravimetric analysis and raised for the subsequent reaction by rotary evaporation. FITC Labeling of CNC-NH2. FITC labeling of CNC-NH2 was carried out in a buffer solution consisting of 240 mL deionized water, 41.1 g sucrose, 0.76 g EGTA, 3.52 g NaCl, and 7.64 g of Na2B4O7· 10 H2O. To this solution, 2 g of FITC was added under stirring and the flask was covered with aluminum foil to exclude light. Next, 50 g of an aqueous suspension of CNC-NH2 of 2 wt % was added under ice-bath cooling. After 4 h, 140 mL of buffer solution was added to dissolve excess FITC that had deposited on the flask wall. The reaction was allowed to proceed overnight in the dark. Then, the reaction mixture was transferred to dialysis tubing and dialyzed against deionized water. When diffusion of excess FITC from the reaction mixture had ceased according to visual inspection of the dialysis water, the suspension was sonicated under ice-bath cooling for 10 min at 40% output and centrifuged for 12 min at 4550g to remove any aggregates. The supernatant was again dialyzed against deionized water until the dialysis water did no longer show FITC UV−vis absorption peaks (∼2 days). The concentration of the obtained aqueous suspension of FITC-CNC, determined by gravimetric analysis, was generally close to 0.1 wt %. Folic Acid Conjugation of FITC-CNCs. Folic acid (0.12 g), EDC (0.054 g), and Sulfo-NHS (0.061 g) were added under stirring to 20 mL PBS. The pH of the reaction mixture was determined with a pH meter (Mettler Toledo S47 SevenMulti pH/conductivity meter with an Inlab 413 pH electrode) and adjusted to 7 by addition of 0.02 N NaOH. Then, 150 g of an aqueous suspension of FITC-CNC of about 0.1 wt % was added and the pH of the reaction mixture adjusted again to 7. The reaction was allowed to proceed for 40 h at room temperature under stirring in the dark. The reaction mixture was processed as described above by dialysis, sonication, centrifugation, and second dialysis until the dialysis water did no longer show folic acid UV−vis absorption peaks (∼2 days). FTIR Spectroscopy. FTIR absorption spectra were recorded with a Thermo Scientific Nicolet 8700 FTIR spectrometer from KBr pellets, prepared as follows, with a resolution of 4 cm−1 and 64 scans per sample. Small amounts of unreacted and reacted CNCs were isolated by freezedrying of aliquots of the respective aqueous suspensions. For KBr pellet preparation, 2 mg of sample was thoroughly ground in a mortar together with 98 mg of anhydrous KBr. The obtained powder was dried in a vacuum oven at 45 °C for several hours and then pressed into a pellet between two stainless steel bolts inserted from opposite ends into a stainless steel nut. FTIR spectra were recorded from the pellets after further drying in a vacuum oven at 45 °C for several hours. UV−vis Spectroscopy. UV−vis absorption spectra were recorded with a Thermo Scientific Evolution 300 UV−vis spectrometer from dilute solutions/suspensions of known concentration between 0.001 and 0.01 wt % in standard cuvettes with an optical path lengths of 1 cm. Atomic Force Microscopy (AFM). AFM images were recorded in ac mode in air with an Asylum Research MFP-3D-Bio atomic force microscope using standard Olympus silicon probes (OMCL-AC160TS) with a nominal tip radius and spring constant of
