Aptamer-Conjugated Apigenin Nanoparticles To Target Colorectal

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Aptamer Conjugated Apigenin Nanoparticles To Target Colorectal Carcinoma: A Promising Safe Alternative of Colorectal Cancer Chemotherapy Debasmita Dutta, Apala Chakraborty, Biswajit Mukherjee, and Sreya Gupta ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00441 • Publication Date (Web): 16 Oct 2018 Downloaded from http://pubs.acs.org on October 18, 2018

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Figure 1. Fourier transform infrared spectroscopic spectra of drug, excipients, their physical mixture, formulation with or without drug and determination of aptamer conjuation to the formulation. Spectra of (A) apigenin; (B) polylactide-co-glycolide (PLGA); (C) polyvinyl alcohol (PVA); (D) PVA+PLGA physical mixture; (E) drug (apigenin)+PVA+PLGA physical mixture; (F) blank formulation (without drug) (G) apigenin-loaded PLGA nanoparticle (ANP); (H) Aptamer conjugated apigenin-loaded PLGA nanoparticle undergone accelerated stability study for 6 months (Apt-ANP 6M ACS) (I) Apt-ANP stored in refrigerated condition (2-8 ºC) for 6 months (J) Pictorial representation of bond stretching in between apigenin and excipients. (K) Confirmation of aptamer conjugation by gel electrophoresis. Aptamer-conjugated nanoparticle (apt-ANP) remained in the well where it was loaded and produced a prominent fluorescence due to intercalation of EtBr with aptamer. Unconjugated ANP did not generate any fluorescence whereas free-aptamer migrated and showed a band parallel to 20 bp size. 101x101mm (300 x 300 DPI)

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Figure 2. Field emission scanning electron microscopic images of (A) Formulation ANP at 20,000×; (B) Formulation ANP at 40,000×; (C) size distribution of apigenin-loaded PLGA nanoparticles (ANP) (D) zeta potential of ANP. (E) size distribution of aptamer conjugated apigenin-PLGA nanoparticles stored storing in refrigerated condition for 6 months (F) zeta potential of Apt-ANP. 635x635mm (96 x 96 DPI)


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Figure 3. Characterization of nanoparticles. (A) Cryo-transmission electron microscopic image of ANP and (B) Cryo-transmission electron microscopic image of Apt-ANP, (C) Three dimensional view of apt-ANP formulaion by Atomic force microscope, (D) Cumulative % drug release from ANP and apt-ANP against time. Data show mean± standard deviation of two different experiments in triplicate. 81x65mm (300 x 300 DPI)



Figure 4. In vitro cellular uptake analysis of nanoformulations by HCT-116 cells. Confocal microscopic images of human colorectal carcinoma cells, HCT-116 after 2 h, 4 h, 6 h and 24 h incubation with FITC (Green) conjugated (A) ANP and (B) Apt-ANP, counter stained with propidium iodide (Red). Flow cytometric assay data demonstrate (C) untreated HCT-116 cells and after 6 h incubation with (D) ANP, (E) Apt-ANP. (F) overlay of untreated, ANP and Apt-ANP treatment of HCT-116 (Red denotes untreated population, green for ANP and blue for (apt-ANP) after 6 h of incubation. 58x73mm (300 x 300 DPI)


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Figure 5. Loss of mitochondrial membrane potential. HCT-116 cells (2.5 x 104/ ml McCoy′s 5a medium) were incubated with IC50concentration of ANP and Apt-ANP for 12 h, 24 h and 48 h. Then the cells were loaded with mitochondrial sensor dye JC-1 (7.5 mM; 15 min) as described in Materials and methods and the analysis revealed a higher percentage of apoptotic cells for Apt-ANP by the shift from blue to green fluorescence in a time-dependent manner. Data is a representative of three different experiments. 39x58mm (300 x 300 DPI)



Figure 6. Modulation of the cellular antioxidants, GSH and mitochondrial enzymes complexes (SDH and NADH oxidase) in HCT-116 cells after treatment with apt-ANP for 12 h, 24 h and 48 h with respect to control value (A) GSH was found to decrease whereas (B) NADH activity increased and (C) SDH level decreased in a time-dependent manner. GSH is expressed as μg/ mg protein. NADH oxidase is represented as nmol oxidase/ min/ mg protein and SDH is represented as mm DCIP reduced/ mg protein. Data demonstrates as mean protein level/ activity ±SEM of three independent experiments (*p < 0.05). 317x81mm (300 x 300 DPI)


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Figure 7. (A) Plasma concentration and (B) colon concentration of apigenin vs time curve represents mean drug concentration at different time intervals after intravenous administration of AP, ANP and Apt-ANP in colorectal cancer mice model. Data represent mean±standard deviation of three different experiments in triplicate, (C) 2 h and (D) 4 h gamma scintigraphy images of mice after administration of 99mTc labeled apigenin nanoparticles (99mTc-ANP) and 99mTc labeled aptamer conjugated apigenin nanoparticles (99mTc-Apt-ANP) through tail vein. 89x89mm (300 x 300 DPI)



Figure 8. Confocal microscopy images of normal and colorectal cancer model colons (cross sections). FITC labelled apt-ANP was injected in normal and colorectal cancer mice and their colons were collected at 2 h and 5 h post injection, sectioned, stained with DAPI and observed under confocal laser microscope at 60X magnifications. 27x20mm (300 x 300 DPI)


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Figure 9. Histopathological morphology of (A) and (B) normal colonic tissue, (C) to (F) showing different grades of cancer progression, (G) and (H) carcinogen treated received apt-ANP, (I) and (J) normal received apt-ANP 74x106mm (300 x 300 DPI)



Scheme 1: Scheme of aptamer modification and conjugation on surface of apigenin nano-particle 254x190mm (96 x 96 DPI)


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Scheme 2: Diagrammatic mechanism of action of apigenin encapsulated PLGA nanoparticles, surface functionalized with aptamer against EpCAM over expressed on colorectal carcinoma cells 40x30mm (300 x 300 DPI)



33x21mm (300 x 300 DPI)


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Aptamer Conjugated Apigenin Nanoparticles To Target Colorectal Carcinoma: A Promising Safe Alternative of Colorectal Cancer Chemotherapy Debasmita Dutta†, Apala Chakraborty†, Biswajit Mukherjee*†, Sreya Gupta‡

†Department

India.

of Pharmaceutical Technology, Jadavpur University, Kolkata-700032, West Bengal,

‡ Department

of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Kolkata, Chunilal Bhawan, 168, Maniktala Main Road, Kolkata – 700054 (West Bengal, India).

*Corresponding Author Prof. (Dr.) Biswajit Mukherjee Department of Pharmaceutical Technology, Jadavpur University, Kolkata-700032, India Telephone: +91-33-2457 2588 Fax: +91-33-2414 6677 Email: [email protected]/ [email protected]

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Abstract Apigenin has gained interest recently among researchers as a potential chemotherapeutic agent in cancer, including colorectal cancer, due to its established antiproliferative activity in vitro. Despite its impressive anticancer activity in vitro, poor water solubility and nonspecific distribution in vivo make it difficult for its emergence as a drug candidate. To overcome these problems, we formulated aptamer-conjugated apigenin-loaded nanoparticle (apt-ANP) to target against overexpressed colorectal cancer cell surface biomarker Epithelial Cell Adhesion Molecule (EpCAM). Aptamerconjugation was conducted on the prepared nanoparticle, characterized (by SEM, TEM and AFM) and evaluated for its antiproliferative activity towards in vitro colon carcinoma cells and in vivo colorectal cancer model. The aptamer-conjugated nanoformulation had an average size about 226 nm, smooth surface, satisfactory drug loading 17.5±1.3% and sustained drug release pattern. Pharmacokinetic profile as well as the biodistribution study demonstrated maximum retention of apt-ANP in colon as compared to free-drug and aptamer-free apigenin-loaded nanoparticle (ANP). Apt-ANP enhanced therapeutic efficacy to colorectal cancer cells whereas minimized off-target cytotoxicity to normal cells. Keywords Apigenin, Nanoformulated drug delivery, Aptamer, Phosphorothioate modification, Colorectal carcinoma, EpCAM receptor. Introduction In last few decades, plant derived natural compounds including vinca alkaloids, epipodophyllo toxins, camptothecins and taxanes have found their way from traditional medicine to mainstream anticancer therapy. Flavonoids, ubiquitously available in vegetables, berries, fruits, tea and chamomile have shown recent promise in this area. Apart from their striking antioxidant properties, flavonoids are important members of plant secondary metabolites chemically related to polyphenols. Consumption of flavonoids has been shown to diminish the occurrence of colorectal neoplasia1, a so-called chemo-preventive effect. A multi-centric case control study reported that consumption of flavones in high amount decreased the incidence of colorectal cancer.2 Among structurally related flavonoids, apigenin has been explored more in last few decades for its enhanced cytotoxicity and higher specificity towards neoplastic cells as compared to normal cells.3 It has been shown through various investigations that apigenin induces apoptosis by arresting G2/M phase of cell cycle in several neoplastic cells including colorectal cancer by modulating some 2 ACS Paragon Plus Environment

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apoptotic proteins namely STAT3, PI3K, ERK, JNK, and AKT.3-7 Despite its impressive anticancer activity, poor water solubility (as per biopharmaceutics classification system it comes under class II)8 and nonspecific tissue distribution, it remains as pharmacological challenges hindering its practical application as an anticancer drug. So far several approaches have been taken to overcome these limitations of apigenin, notably, carbon nano powder adsorbed formulations6, apigenin nanocrystals9 and poly (lactic-co-glycolide) nanoparticles of apigenin for topical use as skin protectant against skin tumor10 and lipid based nanosize capsule.11 However, a common problem of use of nanoparticles is their nonspecific accumulation in cancer tissue as well as normal tissue, reducing their selectivity and dose related efficacy.12 Herein we have developed a FDA approved polymer, poly[lactic-co-glycolic acid] (PLGA)-based apigenin-loaded nanoparticle conjugated to aptamer against EpCAM13, that may circumvent these challenges by improving uptake of the drug as well as specifically targeting them to colorectal cancer cells that express elevated level of EpCAM as a specific biomarker. We chose aptamer mediated targeting over conventional antibodies to improve cellular uptake with almost no immunogenic reaction. Among EpCAM targeting therapeutics, the newest advancement is aptamer conjugation. Aptamer, a synthetic oligonucleotides form three dimensional conformation which directly binds to the target protein, thus they provide the self specific targeting.14-16 Often called as a ‘chemical antibody’, aptamer is advantageous over protein antibodies for its small size, which improves its tissue penetration potential and elicits few to no immunogenic responses. Furthermore, we report modification of the aptamer at 3'-amino group to form a peptide linkage with carboxyl group present on PLGA nanoparticles and replacement of phosphodiester bond with phosphorothioate back bone17 to prevent digestion by DNase in plasma (Scheme 1). However, this thiol modified oligonucleotides aptamer conjugated with nanoparticles are degraded after cellular internalization of nanoparticles with nucleases of Type I and Type II categories.18



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Scheme 1: Scheme of aptamer modification and conjugation on surface of apigenin nano-particle

Designing of aptamer conjugated apigenin nanoparticle and its targeting to colorectal carcinoma is an emerging approach of site specific delivery of drug in the colorectal cancer cells. Colorectal cancer, one of the prevalent types of gastrointestinal neoplasia, is the third most common form of cancer globally.19 It still remains a challenge to combat the disease. This aptamer conjugated nanoparticle when specifically targets the colorectal carcinoma cells, it would help to reduce the dose of apigenin as well as in vivo cytotoxicity. Hence, aptamer conjugated apigenin nanoparticles may be a smart approach to overcome the lacunas associated with free drug and nonspecific apigenin nanoformulation.We prepared aptamer conjugated apigenin nanoformulation and investigated its antiproliferative activity in colorectal carcinoma cells followed by pharmacokinetic activity and in vivo efficacy in mouse model of colorectal cancer (Scheme 2).


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Scheme 2: Diagrammatic mechanism of action of apigenin encapsulated PLGA nanoparticles, surface functionalized with aptamer against EpCAM over expressed on colorectal carcinoma cells

Materials and methods Materials Apigenin was procured from Cayman Chemicals (Michigan, USA). PLGA (MW 4,000-15,000; lactide to glycolide ratio 75:25) was purchased from Sigma-Aldrich Chemicals Pvt.Ltd., Bangalore, India. Polyvinyl alcohol (PVA, MW 1,25,000) was obtained from S.D.fine-chem. Ltd., Mumbai, India. Fluorescein isothiocyanate (FITC) was purchased from Himedia Lab. Pvt. Ltd., Mumbai, India. Acetone, acetonitrile and dichloromethane (DCM) were acquired from E. Merck (India) Ltd., Mumbai, India. Only analytical grade chemicals were used to accomplish the work.



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Mice colorectal cancer model For pharmacokinetic study and in vivo colon cancer model, Swiss albino mice (Swiss albino strain) of approximately 20–25 g weight of both the sex (1:1) were used. The study plan was prepared as per the guideline of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India, and was sanctioned by Animal Ethics Committee (AEC), Jadavpur University before commencement of the experiments. The animals were fed with a standardized food and water. Experimental grouping was done as normal control mice, carcinogen control mice, carcinogen treated mice received free drug, carcinogen treated mice received ANP, carcinogen treated mice received apt-ANP and normal mice received apt-ANP. Each groups contained 6 experimental animals. For the development of colorectal cancer model in mice, 1, 2dimethylhydrazine (DMH) was administered once in a week with a dose of 20 mg/kg body weight by intraperitoneal route for 12 consecutive weeks.20Free-drug treated mice were administered apigenin at a dose of 20 mg/kg body weight.20 Mice treated with nanoformulation were provided a dose of 2.5 mg/kg equivalent by suspending the formulation in water for injection by i.v. route21 once a week for 4 consecutive weeks. Aptamer sequence Aptamer of 48 base sequences targeted against EpCAM which overexpresses on the surface of the colorectal carcinoma cells was synthesized on a 0.05 μmol scale with phosphorothioate backbone modification and undergone HPLC purification (synthesized by Eurofins Genomics India Pvt. Ltd., Bangalore, India). We have selected the following aptamer sequence which is the most explored aptamer by the researchers so far with the lowest Kd value to target EpCAM receptor.15 We have further modified the sequence with replacement of phosphodiester bond by phosphorothioate back bone to protect the aptamer sequence from DNase mediated degradation in plasma. Additionally, 3'-amino modification has been incorporated so that peptide bond could be generated between PLGA of the nanoparticles and aptamer; as shown below: 5'-C*A*C*T*A*C*A*G*A*G*G*T*T*G*C*G*T*C*T*G*T*C*C*C*A*C*G*T*T*G* T*C*A*T*G*G*G*G*G*G*T*T*G*G*C*C*T*G-3'

(3' NH2-C3 modification)

(* indicates Phosphorothioate back bone) Drug-excipients interaction by Fourier transform infrared spectroscopy (FTIR) 6 ACS Paragon Plus Environment

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FTIR spectroscopy was carried out to detect the possible mode of interaction between drug and excipients. Pure apigenin, PLGA, PVA, physical mixture of the excipients with or without apigenin, and the nano-formulations loaded with drug and drug free conditions were inspected under inert atmosphere within the wave number range of 4000-400 cm-1 by FTIR spectrophotometer (Alpha, Bruker, Ettlingen, Germany). KBR pallet was used to acquire FTIR spectrum.22 Aptamer conjugated nanoformulations freshly prepared and stored at 2-8 ºC and at 40 ºC (75% RH) for 6 months were investigated at initial stage and after 6 months qualitatively by this method. Preparation of apigenin encapsulated PLGA nanoparticle (ANP) PLGA nanoparticle with or without apigenin encapsulation was formulated by the method multiple emulsion solvent evaporation, after few definite modifications. 22 Nanoparticles were prepared by 50 mg of PLGA (75:25) dissolved in 2 ml of DCM/Acetone mixture. A ratio 4:1 (v/v) of DCM: Acetone was selected by trial and error to be the best solvent composition for nanoparticle formation with desired physical characterisations. Apigenin (5 mg) was added and dissolved in the same polymeric organic phase. For preparation of nanoparticles, PVA solutions of two different concentration,1.5% (w/v) and 2.5% (w/v) were prepared by dissolving PVA in milliQ water with the help of magnetic stirrer (Remi Equipments, Mumbai, India) with occasional heating. The solution containing both the drug and polymer was emulsified quickly soon after drop-wise addition of required amount of 2.5% w/v PVA solution with continuous homogenization for approximately 3 min under high speed homogenization at 20,000 rpm (using Model T10B, Ultra- Turrax, IKA Laboratory Equipment, Staufen, Germany). The white creamy primary emulsion was then gradually transferred to 1.5% PVA w/v solution under homogenization for about 8 min at 20,000 rpm to form multiple emulsions. The obtainedemulsionthensonicate30 min to segregate the large globular particles and then kept under stirring using magnetic stirrer overnight without heating, for complete removal of organic solvents. Then the particles were centrifuged at 5000 rpm and then supernatant was collected and was further centrifuged at 16000 rpm in cold centrifuge (Hermle refrigerated centrifuge, Siemensstr, Wehingen, Deutschland) to get nanoparticles. The particles were washed thrice with double distilled water to eliminate excess PVA from the surface of the particles and then stored in -20ºC. After that, the frozen sample was lyophilized by Freeze drying technique (Laboratory Freeze Dryer, Instrumentation India, Kolkata, India) to get the nanoparticles.



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Preparation of FITC containing nanoparticles FITC tagged nanoparticles were prepared by the same processes as described above.100 μl of FITC solution in ethanol (0.4% w/v) was added into organic phase containing polymer and drug before homogenization. All the remaining procedure was same as mentioned above.22 Aptamer Conjugation on the surface of nanoparticles, ANP The 3' amino and phosphorothioate back bone modified aptamer was grafted on apigenin nanoformulation,

ANP

using

1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide

(EDC)/

N-

hydroxysuccinimide (NHS) technique. ANP was first suspended in a concentration of 5 mg/ mL of deionized water and then incubated for 30 min at 25 ºC with 200 mM of EDC and 100 mM of NHS. The resulting N-hydroxysuccinimide activated ANP was then rinsed with DNase-RNase free water to eliminate excess EDC/NHS. 0.5 mg/ml of aptamer solution was allowed to denaurate-renaturate by keeping at 85°C for 10 min followed by cooling at ice-water bath for 10 min. The activated ANP was then mixed with denatured-renatured aptamer for at least 6 h under rotation. The covalently linked apt-ANP bioconjugates was rinsed with deionizedwater.23 Agarose gel electrophoresis for aptamer conjugation To confirm the aptamer conjugation, ANPs treated with or without the cross linker (EDC) was subjected to agarose gel electrophoresis. Samples were put inside the well of the gel as following sequences DNA marker, ANP, free-aptamer, apt-ANP. The electrophoresis parameters has been set at 50 V for 160 min and 0.5 mg/ml ethidium bromide was added to visualize the base pair (bp).23

Particle size distribution and zeta potential The average particle size and zeta potential values of ANP and apt-ANP nano formulations were detected by using Malvern Zetasizer Nano-ZS 90 (Malvern Instruments, Malvern, UK) with the help of dynamic light scattering technique. A known quantity of the prepared nano formulation was added and dispersed in Milli-Q water (Millipore Corp., Billerica, MA, USA) followed by vortex and sonication before placing in cuvette for measuring the above mentioned parameters.22 Drug loading (%DL) and entrapment efficiency (%EE) study To determine % drug loading, 2 mg of prepared nano formulation was dissolved in 2 ml of acetonitrile and water (80:20 V/V). The formed solution was kept for maximum 3-4 h in an incubator shaker (Somax Incubator Shaker; Shenjhen Pango Electronic Co. Ltd., Shenzhen, China). 8 ACS Paragon Plus Environment

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The sample then centrifuged at 16,000 rpm for 15 min in a cold centrifuge to separate out the supernatant. The obtained supernatant was then suitably diluted with the same solvent mixture and analysed spectrophotometrically at the corresponding λmax of 340 nm.24 Drug loading was determined as follows: Drug loading (Practical) (%) =Quantity of drug present in nanoparticles × 100/Quantity of nanoparticles taken............(1) Entrapment efficiency was calculated as follows: Entrapment efficiency (%) = Practical Drug loading × 100/ Theoretical Drug loading............(2) Field emission scanning electron microscope (FESEM) Particle morphology was determined by field emissions scanning electron microscope (FESEM) (JEOL JSM‐7600F, Japan). Prepared ANP nano formulation was put over a carbon tape placed on a stub and an at accelerating voltage of 10 kV and 10 mA of current about 5 nm of platinum coating was applied with the help of a platinum coater (JEOL, Tokyo, Japan).25 Cryogenic-transmission electron microscopy (Cryo-TEM)ͦ Internal morphology, of ANP and apt-ANP were confirmed by Cryo-TEM. The nanopartculate images were captured by a camera (BM-Eagle 4 k × 4 k CCD) (FEI Company, Netherlands) and the final pixel size set at 1.89 Å.26, 27

Atomic force microscopy (AFM) The samples were dispersed in Milli-Q water using brief vortexing and sonication before depositing on muscovite ruby mica sheet (ASTM V1 grade ruby mica, Micafab, Chennai, India). One drop of sample was placed in the mica surface and dried in a vacuum dryer. The internal morphology of the particles were analyzed by mode Peak Force QNM (Quantitative Nano Mechanical mapping) in Atomic Force Microscopy (Dimension Icon, Bruker, Karlsruhe, Germany) under ambient conditions using silicon nitride probe having a resonance frequency and a force constant of 150-350 kHz and 0.4 N/m, respectively. The images were captured in a scale of 512 by 512 pixels with a scan area of 2.0 µm × 2.0 µm at the scan speed rate of 0.5 lines/s. Images were analyzed with the help of Pico View 1.12 version software (Agilent Technologies, Santa Clara, CA, USA).24 9 ACS Paragon Plus Environment


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In vitro drug release and drug release kinetics The release mechanism of the drug In vitro was performed at different time points in triplicate by choosing phosphate buffer saline (PBS), pH 7.4,as a media for 60 days. Briefly, nanoparticle (2 mg) was taken accurately in 2 ml micro-centrifuge tube. Drug release media (2 ml) was added in each tube and shaken briefly and the time was considered as zero. The tubes were taken for incubation at 37º±0.5ºC with mild shaking (20 rpm). Samples were taken at different time intervals and then centrifuged for 10 min at 16000 rpm. The supernatant (1 ml) was withdrawn and 1ml PBS was added and the tubes were kept in shaker incubator for next time point. The supernatant was analyzed by UV-Vis spectroscopy with proper dilution, if required, at 340 nm. The drug release data were plotted as percentage drug released (cumulative) vs. time (h).22 In vitro experimental drug release data were tested on various drug release kinetic model, namely, zero-order and first-order kinetics, Higuchi, Hixson–Crowell model and Korsmeyer-Peppas models.22 In vitro study of anti-proliferative activity of apigenin nanoparticles i)

Cell lines and culture The adherent colon carcinoma cells HT-29 and HCT-116 were procured from National Centre for Cell Science, Pune, India. The cells were maintained in RPMI 1640 media (supplemented with 10% FBS, 50 IU/ml penicillin G and 50 µg/ml streptomycin) in a humidified incubator at 37 °C under 5% CO2 environment. Subculturing was carried out on every 72 h and cell viability was tested by trypan blue exclusion method whenever required.28

ii)

Human peripheral blood mononuclear cells (PBMC) isolation process Peripheral blood mononuclear cells (PBMC) were isolated from anticoagulated blood following the method described by Dutta et al.

29

An equal volume of Ficoll-Hypaque

(Histopaque-1077) was added with the anticoagulated blood and centrifuged at 400 x g for 30 min. The PBMC were collected from the interface of two liquids, washed with PBS (0.01 M, pH 7.4) twice and resuspended in RPMI 1640 medium.29


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iii)

Cell viability Peripheral blood mononuclear cells (PBMC) were separated from anticoagulated blood following the method described by Dutta et al.

29

An equal volume of Ficoll-Hypaque

(Histopaque-1077) was added with the anticoagulated blood and centrifuged at 400 x g for 30 min. The PBMC were collected from the interface of two liquids, washed with PBS (0.01 M, pH 7.4) twice and resuspended in RPMI 1640 medium.29 The cytotoxic activity of different nanoformulations dissolved in PBS in presence of very minute amount of dimethyl sulfoxide, DMSO (final DMSO concentration

Aptamer-Conjugated Apigenin Nanoparticles To Target Colorectal

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