Single Layer Assembly of Multifunctional Carboxymethylcellulose on

Subscriber access provided by Drexel University Libraries

Applications and Health

Single Layer Assembly of Multifunctional Carboxymethylcellulose on Graphene Oxide Nanoparticles for Improving in Vivo Curcumin Delivery into Tumor Cells Foozie Sahne, Maedeh Mohammadi, and Ghasem D. Najafpour ACS Biomater. Sci. Eng., Just Accepted Manuscript • Publication Date (Web): 25 Mar 2019 Downloaded from http://pubs.acs.org on March 25, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Biomaterials Science & Engineering

Single Layer Assembly of Multifunctional Carboxymethylcellulose on Graphene Oxide Nanoparticles for Improving in Vivo Curcumin Delivery into Tumor Cells Foozie Sahne†, Maedeh Mohammadi†*, Ghasem D. Najafpour† †Biotechnology

Research Laboratory, Faculty of Chemical Engineering, Babol Noushirvani

University of Technology, Shariati Avenue, 47148, Babol, Iran * Corresponding author: Phone: +98 11 32334204; Fax: +98 11 32367975 E-mail address: [email protected] Abstract Nano-drug delivery systems are considered as promising therapeutic platforms to convey drugs to tumor cells. In this study, a single layer of carboxymethylcellulose (CMC) and poly Nvinylpyrrolidone (PVP) was cross linked through disulfide bond and deposited on graphene oxide nanoparticles (GO NPs) using layer-by-layer technique. Overexpression of folate receptors on tumor cells is a great hallmark for drug delivery systems; though the NPs were functionalized by monoclonal folic acid antibody (FA) using polyethylene glycol (PEG) as linker. The mean diameter of synthesized nanoparticles was 60 nm. Curcumin was encapsulated within CMC layer with high encapsulation capacity of 94%. In vitro investigation showed 87% curcumin release at simulated tumor environment. Curcumin loaded FA modified CMC/PVP GO NPs showed high inhibition of 76 and 81% against Saos2 and MCF7 cell lines in vitro. In vivo investigations on 4T1 bearing breast cancer mice model exhibited 76% antitumor efficiency via active targeting mechanism of folate mediated transport without any significant side effect. Immunohistochemistry and immunofluorescence analyses showed enhanced anti-angiogenesis, apoptosis and tumor growth inhibition. Keywords: Graphene oxide; curcumin; folic acid antibody; stimuli-responsive; tumor targeting

1 ACS Paragon Plus Environment

ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Introduction Nowadays, genesis of multiple life-threatening cancers has increased the mortality rate. Recent studies have indicated that cancer treatment strategies are turning towards use of natural bioactive compounds, yet the rapid clearance and enzymatic cleavage of these compounds are the major barriers for their biopharmaceutical applications. Advanced drug delivery systems have found ways to address part of these issues; nanobiotechnology as an emerging field of research in this area has developed its branches in nano-drug delivery, diagnosis, bio-imaging and medicament for treatment of chronic cancers.1 Particularly, usage of nanocarriers is a strategy for improving the bioavailability and water solubility of bioactive compounds. Furthermore, the well systemic biodistribution at targeted sites without any nonspecific accumulation offers significant opportunities for special medical applications of nanocarriers.2 Curcumin is an active pseudo symmetric polyphenolic compound and numerous evidences have shown the potency of curcumin as anti-oxidant and anti-cancer agent.3 Curcumin is considered as a highly pleiotropic and functionally labile molecule which can affect the biological activity of multiple signaling molecules such as inflammatory molecules,4 cell proteins, histone acetyltransferase (HATs), receptors, DNA, RNA and metal ions.5-9 Despite numerous curative properties of curcumin, its medicinal application is limited by rapid metabolism, poor aqueous solubility (11 ng/ml) and systemic bioavailability.10 These issues would be addressable with advanced pharmaceutical formulation. Accordingly, drug delivery systems consisting of therapeutic drug and molecularly engineered nanocarriers are outperforming the conventional one-size-fits-all nanoformulation approaches .11-12 It is important to point out that the systemic administration and biological behavior of nanostructures are major factors for efficient tumor microenvironment delivery. Blood-protein interaction, systemic 2 ACS Paragon Plus Environment

Page 2 of 40

Page 3 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


circulation, tumor cell interaction and physico-chemical characteristics of nanostructures can influence the pharmacokinetics and biological processes. Recent insights into graphene oxide as nanocarrier has shed light on the field of nanoformulation in order to increase the bioavailability of bioactive compounds.13-16 Specific targeted delivery of drugs using graphene oxide can be developed by its functionalization with carbohydrates, nucleic acids, aptamers, antibodies and etc. The graphene based materials render more permeability and enhance cellular internalization through “piercing effect”.17-18 Nasrollahi et al,19 found functionalized graphene oxide nanocarriers as potential drug delivery systems into MCF7 cell lines. Gong et al,20 synthesized fluorinated graphene nanoparticles, modified with folic acid, as targeted endocytose into cancer cells. Herein, a facile and versatile surface modification was conducted on graphene oxide nanoparticles (GO NPs) for targeted binding to tumor cells in vivo. The NPs were covered by a single layer of carboxymethyl cellulose (CMC). In order to prepare stimuli- responsive NPs, poly (N-vinyl pirrolidone), (PVP) was synthesized and immobilized on the NP surface which induces redox-responsive disulfide linkage by conjugating to thiolated CMC layer. Recently, stimuliresponsive polymers (SRP) have attracted considerable interest in the field of drug delivery systems. The SRP devices can respond to endogenous stimuli like pH variation,21 glutathione concentration, certain enzymes and reactive oxygen species or extracorporeal stimuli such as light 22, magnetic and electric field and temperature.1, 10, 23 In this work, PVP with amine and thiol-reactive end groups was synthesized using reversible addition-fragmentation chain transfer (RAFT) polymerization. The stimuli-responsive bridge on the CMC/PVP GO NPs was prone to cleavage by high tumor intracellular concentration of glutathione (~2-10 mM) compared to its



extracellular concentration (~2-10 µM).23 The glutathione responsive characteristics of CMC/PVP GO NPs was successfully explored in simulated cancer condition. Monoclonal antibody functionalized nanocarriers represent promising modality for cancer treatment. Antibodies bind precisely to the targeted receptors of interest. Folate receptors (FRs), especially FRα, are highly expressed on the cancer cells surface to resolve demand of folate deficiency in dividing cancer cells, though FRs are considered as molecular target for cancer therapeutic treatment. Folic acid antibody (FA) binds perpendicularly to globular αhelices structure of FR through hydrogen and hydrophobic interactions.24 Then, nanocarriers can easily enter the tumor cells through phagocytosis. To demonstrate the applicability of functionalization, in this study, FA were coupled with bifunctional N-Hydroxysuccinimide (NHS)-PEG through succinimid ester group and attached to CMC/PVP GO NPs surface. Adsorption of blood proteins such as immunoglobulins and complement proteins onto nanocarriers surface results in ‘protein corona’; this phenomenon facilitates renal clearance by mononuclear phagocyte and hinders targeting efficiency.25 So far, PEGylation of nanocarriers has been used to reduce protein adsorption and prolong the blood circulation. Moreover, incorporation of PEG could prevent drug from biological degradation, enzymatic cleavage and uncontrolled leakage. In this study, a novel polymer synthesis method was developed and the synthesized polymer was used as a potential nano-drug delivery system to deliver anti-cancer drugs into tumor cells. A single layer of thiol-acetylated CMC was assembled on GO NPs in order to increase drug loading efficiency which was further conjugated to PVP to induce redoxresponsive linkage. In addition, targeting characteristics of the NPs were addressed by FA-PEG ligand functionalization. Here, the curcumin (Cur) extracted from turmeric, as we did in our


Page 4 of 40

Page 5 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


previous study,26 was selected as hydrophobic drug model with potent anti-cancer properties. The cytotoxic effect of multifunctional Cur-FA-CMC/PVP GO NPs for delivering curcumin into malignant tumor cells was investigated in vitro and in vivo. The schemes of evolution of Cur-FACMC/PVP GO NPs and therapeutic mechanism in the tumor cells are shown in Scheme S1 in Supplementary Material. Experimental Procedures Materials The materials used to develop the Cur-FA-CMC/PVP GO NPs are provided in Sec. S1.1 in Supplementary Material. Preparation of Cur-FA-CMC/PVP GO NPs Graphene Oxide Synthesis GO NPs were synthesized using modified Hummer method.27-28 After synthesis, the mixture was centrifuged at 13000 rpm for 30 min; the precipitate was exfoliated and ultrasonicated for 15 times with 5 wt% H2SO4, followed by centrifugation and collection of the supernatant. The GO mixture was treated for another 15 times with 10% HCl aqueous solution to remove residual metal ions, then dialyzed against deionized water for 2 days for neutralization. The pelleted GO was obtained by centrifugation at 13500 and lyophilization. Acetylation of CMC Acetylation of CMC was conducted by the method reported by Namikoshi et al.29 Acetylated CMC was obtained by lyophilization and acetylation was confirmed by FTIR analysis. CMC thiolation was performed according to the method adopted from Zelikin et al.30 Briefly, 50 mg/ml solution of acetylated CMC was prepared in phosphate buffer saline (PBS 0.1 M, pH 7.2) with 2 ml of DMSO. Then, 70 mg of EDC and 45 mg of NHS were added to the mixture and



stirred for 30 min. Thiol groups were synthesized by adding 75 mg of cystamine dihydrochloride through carbodiimide coupling for 12 h. The resulting polymer was dialyzed against water and lyophilized to obtain fine powder. Thiolation was verified using FTIR analysis. PVP polymerization Thiol and amine modified PVP were synthesized by RAFT polymerization using phthalimidomethyl xanthate as RAFT agent31. The cleavage of thiocarbonylthio to thiol end groups in RAFT-synthesized PVP was accomplished using sodium borohydrate; the primary amine end group was obtained through hydrazinolysis of terminal phthalimide in RAFTsynthesized PVP. The presence of thiol groups in the synthesized PVP was confirmed by mixing the PVP with excess 0.5 M Ellman’s reagent for 1 h in 0.1 M PBS, pH 7.4 and cystamine standard calibration curve. The polymer was recovered by lyophilization and analyzed using HNMR. The redox-responsive disulfide crosslinked CMC/PVP GO NPs were assembled using layer-by-layer deposition of CMC and polymerized PVP.31 For this purpose, 100 ml of 1 mg/ml solution of GO was prepared in sodium acetate buffer (pH 4), followed by addition of 100 ml of 1 mg/ml of CMC solution and adsorption was allowed to proceed for 2 h at constant stirring at room temperature. Then, 100 ml of curcumin solution of 1 mg/ml in acetate buffer with 5 ml DMSO was added to the solution followed by addition of 70 mg of EDC and 20 mg of NHS; the reaction was proceeded for 12 h. Consequently, 1 mg/ml of amine functionalized PVP solution in acetate buffer (pH 4) was adsorbed as outermost layer. The solution was centrifuged and washed by fresh buffer several times. In this study, a single layer of CMC/PVP was assembled on GO NPs. Upon completion of the layer assembly, in order to transform thiol groups into disulfide linkage in CMC layer, 2.5 mM chloramine-T in MES buffer solution (pH 6) was added to the


Page 6 of 40

Page 7 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


solution. Then, the CMC/PVP GO NPs were washed with PBS (pH 7.2) to remove any remained hydrogen-bonded PVP layer.32 The conjugation efficiency of outermost PVP layer was determined by Fluorescein isothiocyanate (FITC) labeling of the amine end groups and subjecting the sample to flow cytometry analysis. Monoclonal FA were coupled with linear bifunctional PEG through NHS activated end group. First of all, FA was dispersed in 50 ml of PBS, then combined with 5 ml of 1 mg/ml FITC in DMSO and stirred overnight. The FITC-labeled FA was separated from free FITC via rinsing the sample with fresh buffer for several times. Then, 20 ml of 20 mM PEG-NHS solution was prepared in PBS and subsequently added to 20 ml of 1 mg/ml FITC-labeled FA solution. The mixture was stirred for 1 h at 4 oC, then 20 ml of antibody solution was incubated with CurCMC/PVP GO NPs in PBS for 1 h at 4 oC. As the process completed, FA functionalized CurCMC/PVP GO NPs (denoted as Cur-FA-CMC/PVP GO NPs) were washed with PBS and quantitatively characterized by flow cytometry for antibody conjugation.32 The overall synthesis procedure of nanoparticles and their therapeutic mechanism within the body is represented in Figure 1.The developed Cur-FA-CMC/PVP GO NPs were subjected to several analyses and characterizations as described in Sec. S1.2. The curcumin loading capacity and encapsulation efficiency were calculated as described in Sec. S1.3. The curcumin release profile from Cur-FACMC/PVP GO NPs was studied in vitro; the method is described in Sec. S1.4.



(a)

(b) Figure 1: Schematic representation of (a) Cur-FA-CMC/PVP GO NPs evolution; GO NPs were functionalized using CMC, then curcumin was encapsulated within the CMC layer which was consequently coated by PVP through disulfide bridge; at the end, FA was conjugated on the surface using PEG as cross linker and (b) in vivo therapeutic mechanism of the developed Cur-FA-CMC/PVP GO NPs which includes tumor cells targeting using FA, cell internalization through folate receptor and drug release in cytoplasm of tumor cells in response to high glutathione concentration 8 ACS Paragon Plus Environment

Page 8 of 40

Page 9 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Cellular Studies Cell toxicity assay of Cur-FA-CMC/PVP GO NPs was conducted against MCF7 (human breast adenocarcinoma) and Saos2 (human bone osteosarcoma adenocarcinoma) cells line. The cells were obtained from Pasteur Institute, Tehran, Iran and cultured in RPMI medium which is used for growth of human lymphoid and leucocytus cells. The medium was supplemented with a great deal of PBS, 10% BSA, and 1% penicillin/streptomycin. The cells were cultured at 37 °C under humidified 5% carbon dioxide atmosphere in an incubator and media were exchanged every 2 days. Six different concentrations of Cur-FA-CMC/PVP GO NPs were prepared in DMEM (the medium contained high concentration of vitamin, amino acid and glucose) and RPMI media with 10% BSA and 1% penicillin/streptomycin.33-34 Determination of cell viability and metabolic activity was based on the protocol described in Sec. S1.5. Animal Studies The 4T1 bearing tumor model BALB/C mice (3-4 weeks) were housed under pathogen-free conditions. Animals had access to sterilized food pellets and distilled water at 12 h light/dark cycle. The animal experimental procedures were approved by the Institutional Animal Care and Use Committee. The mice bearing subcutaneous 4T1 breast cancer models were given the FITC-labeled Cur-FA-CMC/PVP GO NPs via tail vein injection. The mice were then subjected to imaging after treatment during 24 h. After treatment, the mice were sacrificed and tumor tissue and major organs including heart, liver, spleen, lung and kidney were collected and subjected to fluorescence imaging. FITC-labeled Cur-FA-CMC/PVP GO NPs (200 µL, 4 mg/kg curcumin equivalent dose) were injected i.v. into mice bearing subcutaneous 4T1 breast cancer to study



Page 10 of 40

drug uptake in tumor cells. The mice were sacrificed after treatment and tumors were dissected for further immunohistochemistry analysis. For antitumor efficiency assay, the mice bearing 4T1 breast cancer model were randomly divided into 3 groups (n=4): control group which received PBS, Cur-free- FA-CMC/PVP GO NPs treated group (200 µL) and Cur-FA-CMC/PVP GO NPs treated group (200 µL, 4 mg/kg curcumin equivalent dose). The mice were treated via tail vein injection every day after the tumor volume had reached 50-100 mm3 for 3 weeks. The tumor size and body weight of mice were monitored every day. The tumor volume was calculated using the following formula: 𝑉=

𝜋 × 𝑙𝑎𝑟𝑔𝑒 𝑑𝑖𝑎𝑚𝑒𝑡𝑒𝑟 × 𝑠𝑚𝑎𝑙𝑙 𝑑𝑖𝑎𝑚𝑒𝑡𝑒𝑟 6

(1)

After 3 weeks of treatment, the tumor was collected for examination of apoptosis, tumor blood vessels and inflammatory response using immunohistochemical and immunofluorescence analysis. First of all, cryosection of tumor cells carried out as follows. The tumors were excreted and embedded into liquid nitrogen, then tumors were dissected into several 7 µm sections. For immunofluorescence studies, briefly, the tumor slices were incubated with primary antibody in incubation buffer after blocking with goat serum for 1 h at 37 °C. After washing with buffer, slices were incubated with NorthernLights secondary antibodies for 1 h at room temperature as protocol, followed by washing with PBS. The nuclei of cells were stained with DAPI (4',6diamidino-2-phenylindole). For immunohistochemistry studies, briefly, the cells were incubated with related antigen retrieval buffer for 15 min at 95 °C, then washed three times with PBS. After blocking with goat serum for 1 h at 37 °C, the cells were incubated with secondary antibody overnight at 4 °C. The secondary antibody solution was decanted and the cells were washed with PBS. To detect proliferation of the tumor cells, the sections were incubated with pan-cytokeratin antibodies at 4 10 ACS Paragon Plus Environment

Page 11 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


°C overnight. For tumor blood vessels, the sections were incubated with CD31 and CD34 antibodies. Also, for anti-inflammatory response of tumors, the sections were incubated with CD45 as immune cells detector of tumor cells as the protocols. Lastly, the major organs were dissected for hematoxylin and eosin (H&E) staining and assessing the side effect of drug on major organs. The nuclei of cells were stained with hematoxylin and cytoplasm was stained by eosin as the protocols. Statistical Analysis Statistical analyses were carried out using OriginPro 2016 software. All the experiments were conducted in triplicate and results were reported as mean value ± SD. Data were analyzed using one-way ANOVA and one sample t-test analysis; statistical significant differences were at *p

Single Layer Assembly of Multifunctional Carboxymethylcellulose on

Recommend Documents