Engineered Cellular Uptake and Controlled Drug Delivery Using Two

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Engineered Cellular Uptake and Controlled Drug Delivery Using Two Dimensional Nanoparticle and Polymer for Cancer Treatment Sudipta Senapati, Rashmi Shukla, Arun Kumar Mahanta, Dipak Rana, Pralay Maiti, and Yamini B Tripathi Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b01119 • Publication Date (Web): 04 Jan 2018 Downloaded from http://pubs.acs.org on January 4, 2018

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Molecular Pharmaceutics

Engineered Cellular Uptake and Controlled Drug Delivery Using Two Dimensional Nanoparticle and Polymer for Cancer Treatment

Sudipta Senapati,1 Rashmi Shukla,2 Yamini Bhusan Tripathi,2 Arun Kumar Mahanta,1 Dipak Rana3 and Pralay Maiti

1

*1

School of Materials Science and Technology, Indian Institute of Technology (Banaras Hindu University), Varanasi 221 005, India

2

Department of Medicinal Chemistry, Institute of Medical Science, Banaras Hindu University, Varanasi 221 005, India

3

Industrial Membrane Research Institute, Department of Chemical and Biological Engineering, University of Ottawa, 161 Louis Pasteur St., Ottawa, ON, Canada KIN 6N5

*

Correspondence should be made to Pralay Maiti ([email protected])

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Abstract: Two major problems in chemotherapy, poor bioavailability of hydrophobic anti-cancer drug and its adverse side effects causing nausea, are taken into accounts by developing sustained drug release vehicle along with enhanced bioavailability using two-dimensional layered double hydroxides (LDHs) with appropriate surface charge and its subsequent embedment in polymer matrix. A model hydrophobic anti-cancer drug, raloxifene hydrochloride (RH), is intercalated into a series of zinc iron LDHs with varying anion charge densities using ion exchange technique. To achieve significant sustained delivery, drug intercalated LDH is embedded in poly(ε-caprolactone) (PCL) matrix to develop intravenous administration and to improve the therapeutic index of the drug. Cause of sustained release is visualized from the strong interaction between LDH and drug, as measured through spectroscopic techniques like X-ray photoelectron spectroscopy, infrared, UVvisible spectroscopy and thermal measurement (depression of melting temperature and considerable reduction in heat of fusion) using differential scanning calorimeter, followed by delayed diffusion of drug from polymer matrix. Interestingly, polymer nanohybrid exhibits long term and excellent in vitro antitumor efficacy as opposed to pure drug or drug intercalated LDH or only drug embedded PCL (conventional drug delivery vehicle) as evident from cell viability and cell adhesion experiments prompting a model depicting greater killing efficiency (cellular uptake) of delivery vehicle (polymer nanohybrid) controlled by its better cell adhesion as noticed through cellular uptake after tagging of fluorescence rhodamine B separately to drug and LDH. In vivo studies also confirm the sustained release of drug in the blood stream of albino rats using polymer nanohybrid (novel drug delivery vehicle) along with healthy lever vis-à-vis burst release using pure drug / drug intercalated LDHs with considerable damaged lever. Key words: Layered double hydroxide; controlled drug delivery; cellular uptake; in vivo drug release.


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Introduction Cancer, already the leading cause of death globally, is set to become a major cause of morbidity and mortality in the next few decades, irrespective of the human development index of the country.1,2 Conventional chemotherapy is the most commonly used technique for cancer treatment and is based on the killing of cancer cells or inhibition of their growth rate. However, during the process of killing cancer cells, chemotherapeutic drugs also damage healthy tissues, causing severe unintended and undesirable side effects e.g. loss of appetite and nausea. A major reason for this high mortality rate of the cancer patient is due to the severe adverse effects induced by the chemotherapeutic drugs on healthy tissues and organs.3-5 Therefore, controlled and targeted delivery of the chemotherapeutic drugs at the site of action is necessary to maximize the killing effect during the tumor growth phase and to avoid drug exposure to healthy adjacent cells, thereby reducing drug toxicity. The drugs used in chemotherapy often suffer from critical obstacles like poor solubility, drug instability, poor cellular uptake and have severe adverse effects on normal tissues.4,5 To improve therapeutic efficacy, high payload of drug, protect the drug from degradation, facilitate cellular uptake and reduce toxicity and frequency of drug administration, various drug delivery systems have been developed over last few decades. Although considerable progress has been made in cancer therapy, however the complete eradication of cancer and chemotherapy induced adverse side effects remains one of the greatest challenges at the present time. The designing of new drug delivery systems are constantly being tested and improved to enhance the efficacy, selectivity and total effect of the anti-neoplastic drugs while minimizing its adverse side effects. Therefore, controlled and prolonged release of drugs in the therapeutic range in vivo is an important theme to be concerned. To address these problems, the application of nanotechnology for drug delivery is getting considerable attention in recent years to bring new hope for chemotherapy.6-8 Layered double



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hydroxide nanoparticles are one of the best nanocarrier due to their excellent anion exchange capacity, excellent biocompatibility, low toxicity, high drug loading efficacy, full protection for the loaded drugs, low cost, ease of preparation, biodegradability in the cellular cytoplasm (pH = 4–6), biocompatibility, pH dependent stability, excellent endosome escape and moreover drug release rate can be tuned by changing the interlayer anions.9-11 LDHs are a family of anionic clay materials, commonly defined by the general formula [MII

1−xM

III

x

(OH) 2]x+ (An−)x/n.yH2O, where MII is a

divalent metal ion, such as Mg2+, Ca2+, Ni2+, Zn2+, etc., MIII is a trivalent metal ion, such as Al3+, Fe3+, Co3+, Cr3+ etc. and An− is an anion, such as Cl−, NO3 −, CO32−, etc.10 LDHs consist of hydroxide layers of divalent metal cation such as Mg2+, Zn2+, Ni2+, Ca2+, etc., with trivalent metal cation isomorphically substituted to give the layers a net positive charge. This extra positive charge is counter balanced by interlayer hydrated exchangeable gallery anions, such as Cl−, NO3 −, CO32− etc. Due to its excellent anion exchange capacity, any negatively charged functional biomolecules can easily be intercalated into LDH interlayer spacing. These positively charged LDHs can easily penetrate the cellular membrane and therefore, has the potential to serve as efficient intercellular delivery vehicle for hydrophobic, membrane-impermeable therapeutic molecules.12-14 Hence, most of the anionic biomolecules such as amino acids,15 vitamins,16 pesticides,17 drugs18,19 and DNA12,20 are encapsulated into LDH layers in order to form molecular reservoirs for their controlled delivery. However, these LDH-drug nanohybrids have very fast in vitro release profile, almost 100% drug release occurs within couple of hours.18,19 LDH-drug nanohybrids also have very fast in vivo release profile. Choy et al. observed that concentration of 5-fluorouracil (5-FU) in plasma after the administration of 5-FU-LDH nanohybrid follows a rapid declining profile and almost disappeared from the blood stream within 8 h of post injection.21 Again the surface positive charge of LDHs can interfere with negatively charged plasma proteins and adversely influences their pharmacokinetic behavior and reduces their blood residence time. Biodegradable polymers are attractive materials


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from the standpoint of biocompatibility, degradability, bioactivity and have the ability to deliver the drug in a sustained manner. Amongst them, poly(ε-caprolactone) (PCL) is one of the biomaterials known for its biodegradability, enzymatic cleavage at physiological conditions generating non-toxic degradation by-products, biocompatibility, chemical and thermal stability, good mechanical properties and permeability required for wide range of applications such as implantable devices and has the potential to act as sustained drug delivery vehicle.22,23 However, PCL is a hydrophobic material, leading to weak cell-material interactions and as a consequence has poor cell adhesion properties.24,25 Cellular uptake process begins after the initial adhesion of the materials to the cell through the interactions with the lipids, proteins and other components of the cell membrane. The weaker the cell-material interaction (cell adhesion), lower will be the cellular uptake efficacy. Hence, nanohybrid based delivery system is expected to combine the advantages of LDH nanoparticle and polymer vector while overcome their shortcomings. In the present work, a novel nanohybrid drug delivery vehicle has been designed by embedding drug intercalated layered double hydroxide in PCL matrix to improve the therapeutic efficacy of the hydrophobic anticancer drugs by enhancing bioavailability, in vitro and in vivo prolonged and sustained drug release profile, superior cellular uptake, better cancer cell killing efficacy while reducing in vivo adverse side effects to other body parts through hydrophilic hydrophobic balance. Nanohybrid has the ability to suppress the initial burst release of drug and prolong the release period more than 4 days both in vitro and in vivo. In vitro anti-tumor efficiency and enhanced cellular uptake as compared to pure drug or direct drug embedded polymer has been demonstrated and a model has been proposed for greater cellular uptake through its better cell adhesion from hydrophilic hydrophobic balance of the developed material. Animal model and invivo measurements confirm the efficacy of this novel drug carrier through reduced adverse side effects and better killing efficiency of cancer cells for much improved patient compliances.



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Materials and Methods: Materials: Poly(ε-caprolactone) (PCL; Mn ∼80,000) and the anti-tumor drug, Raloxifene hydrochloride (RH), were purchased from Sigma-Aldrich, USA. Zn(NO3)2·6H2O, Fe(NO3)3·9H2O, NaNO3, Na2CO3, and Na3PO4 were obtained from Merck India Ltd. (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide), MTT), Dulbecco’s Modified Eagle Medium (DMEM), penicillin/streptomycin and fetal bovine serum were obtained from Himedia, India. Dimethyl sulphoxide (DMSO) and paraformaldehyde were purchased from Merck India Ltd. Synthesis of 2-D layered double hydroxide (LDHs): The Zn-Fe-LDHs were synthesized by using coprecipitation method according to our previous study.10 In brief, 100 mL mixed solution of 0.5 M Zn(NO3)2·6H2O and 0.25 M Fe(NO3)3·9H2O was added drop wise under N2 atmosphere into 100 ml of 1.5 M NaNO3 or Na2CO3, or Na3PO4 separately and the solutions were stirred vigorously during mixing. 1 M NaOH was added drop-wise under constant stirring to adjust the pH to ~9.5. The whole solution was stirred continuously at 60 οC for 14 h. The appearances of white gelatinous precipitate indicates the formation of Zn-Fe-LDHs having three different intercalated anions of NO3−LDH, CO3−LDH and PO4−LDH and are abbreviated as ZN, ZC and ZP, respectively. The precipitates were recovered by centrifugation (5000 rpm, RT) and were washed thoroughly with deionized water followed by drying in air oven at 60 oC for 24 h. Intercalation of drug (raloxifene) into LDHs: The intercalation of raloxifene into LDHs was carried out through anion exchange method. Typically, to prepare RH intercalated

ZN (NO3-LDH), 1 g ZN was dispersed in 100 ml of

deionized water and was placed in a three-neck round bottom flask followed by degassing through N2 gas purging and pH was adjusted to 8 by using 1 M NaOH solution. 0.15 g of the drug was then added into the solution and was stirred vigorously at 50 oC for 18 h. Then, the precipitate was


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filtered, washed with water and dried at 50 oC. The RH intercalated LDHs are abbreviated as ZN-R, ZC-R and ZP-R using NO3-LDH (ZN), CO3-LDH (ZC) and PO4-LDH (ZP), respectively. Preparation of drug embedded polymer nanohybrid: In order to prepare drug embedded polymer nanohybrid, ZN‒R first sonicated in dichloromethane (DCM) to achieve a good dispersion. PCL was dissolved in DCM separately. Dispersion of ZN‒R in DCM was added to the PCL solution (25% ZN‒R w/w with respect to PCL) and then the whole solution was stirred for 3 h to ensure proper mixing. This mixture solution was allowed 24 h in a fume hood for solvent evaporation and then vacuum dried at room temperature for an additional 24 h. We designate this PCL and ZN‒R nanohybrid as PN‒R. For the control, drug was embedded in pure polymer (PCL) in the similar way and drug embedded polymer is termed as PCL‒RH. Characterization: Physicochemical characterization: The surface morphology of the samples was investigated by using a SEM (SUPRATM 40, ZEISS) and an AFM (NT-MDT, Russia). TEM images were recorded on a JEOL 2010 transmission electron microscope operating at 200 kV. The average particle size (z-average size), its distribution and ζ‒potential of the samples were determined by using a nanoparticle analyzer SZ-100 instrument (Horiba Scientific, Japan) based on the principle of dynamic light scattering at 25 °C. FTIR spectra were measured in the transmittance mode at room temperature from 400-4000 cm-1 on a Thermo Scientific FTIR (NICOLET-6700) with a resolution of 2 cm-1 using the KBr pellet method. X-ray powder diffraction patterns were recorded by using an advance wide-angle X-ray diffractometer with Cu-Kα radiation and a graphite monochromator (wavelength, = 0.154 nm, Rigaku, MiniFlex-600, Japan) at the scanning rate of 3o min-1. UVVisible measurements were carried out using Jasco-V-650 spectrophotometer, Japan, operating in the spectral range of 190 - 1100 nm. The melting point and heat of fusion of the materials were



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measured using differential scanning calorimeter (DSC, Mettler 832) at a scan rate of 10o min-1. The thermogravimetric analysis (TGA) was carried out using a Mettler-Toledo TG/DTA. The surface chemistry and oxidation states (Zn and Fe) of the samples were analyzed using a VSW made photoelectron spectrometer under ultra high vacuum (~ 4.4×10−10 Torr). Binding energies in all XPS spectra were calibrated using C1s peak (284.6 eV). Contact angle measurement was done to estimate the hydrophilicity of samples using a Kruss F-100 tensiometer with three specimens of each sample in the form of thin strips (1 × 10 × 15 mm3) in deionized water. Determination of drug loading: UV-Vis spectroscopic technique was employed to determine the percentage of drug loading in various samples.10 The percentage of drug loading was measured to be 15.4, 17.2, 6.3 and 7.5 for ZN-R, ZP-R, PCL-RH and PN-R, respectively. In vitro Drug release study: The drug release studies were performed at 37 oC after suspending drug intercalated LDH/nanohybrid in 100 ml phosphate buffer saline of pH 7.4. At predetermined time intervals, 1 ml of aliquots were withdrawn and replaced by 1 ml fresh PBS to maintain the sink condition. The concentration of the released drug was determined through UV absorption at λmax = 295 nm. Further, to understand the release kinetics of the drug from the matrix, several kinetic models were used (details given in the Supplementary document) and fitted with the in vitro drug release data. Cell Culture and maintenance: Human cervical carcinoma (HeLa) cells were purchased from National Centre for Cell Science (NCCS), Pune, India. HeLa cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) medium containing 10% heat inactivated fetal bovine serum (FBS), 100 U/ml penicillin and 100 µg/ml streptomycin. The temperature of the culture was maintained at 37 oC in a CO2 incubator with 5% CO2 supply. Biocompatibility


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Cell viability: Cell viability of pure LDHs, pure polymer, pure drug, drug intercalated LDHs (D‒LDH) and polymer D‒LDHs nanohybrids (PN‒R) were performed through the MTT assay against HeLa and NIH-3T3 cells.26,27 In brief, 1×105 cm-2 HeLa/3T3 cells were seeded onto the 96well culture plates in DMEM. After 24 h of culture, the medium in the wells was replaced with the fresh DMEM containing the samples in the concentrations range 10 - 100 µg/ml and were incubated for 24, 48 and 72 h time intervals. After incubation, 100 µl of MTT dye solution (0.5 mg/ml in PBS, pH 7.4) was added to each well by replacing the media containing samples and was again incubated for another 4 h. The absorbance was measured at 570 nm using a microplate reader. Cell viability was calculated by the following equation: % cell viability =

OD of test ×100 OD of control

where, C is the optical density of ‘control’ representing HeLa/3T3 cells incubated in medium alone and T is the optical density of test specimen representing HeLa/3T3 cells treated with the corresponding extracts. Fluorescence imaging: HeLa/3T3 cells were seeded in 24 well plate on cover slips as described earlier and 1×104 cm-2 cells were treated with 80 µg/mL concentration of pristine LDH, drug intercalated LDHs and equivalent amount of pure drug (RH) and were incubated at 37 oC in CO2 incubator. Cells on cover slips were stained with 100 µg/ml acridine orange and 100 µg/ml ethidium bromide. The images were captured through fluorescence microscope. Cell adhesion: The cell adhesion behavior of pure LDH, pristine PCL and PCL-LDH nanohybrids was evaluated through a modified crystal violet staining assay protocol28,29 and was observed using phase contrast microscope. In brief, 1×104 cm‒2 HeLa/3T3 cells were seeded on the sample surface and incubated for 12 h. Then the specimens were washed with PBS twice to remove the unattached



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cells and then the attached cells were fixed with 4% paraformaldehyde solution for 20 min. Cell permeabilization was carried out with 20% methanol for 20 min after PBS washing. The attached cells were then stained using 0.2% crystal violet aqueous solution (Himedia, India) for 20 min. Excess stains were removed by gentle washes in PBS twice followed by elution of the residual crystal violet with 10% acetic acid. Optical density (OD) of the eluted solution was measured using a microplate analyzer (BioTek, USA) at a wavelength of 570 nm, with the background absorbance value measured at 650 nm. The optical density values thus obtained were correlated directly with the number of attached cells in the sample. Furthermore, cells adhesion behaviors were observed using a phase contrast microscope (Leica, Germany) after fixing the cells with 4% paraformaldehyde solution followed by washing with PBS. Cellular uptake studies - Rhodamine-B (RdB) labeling of LDH and RH: LDH particle (ZN) surfaces were first modified by grafting the amino silane ((3-aminopropyl)triethoxysilane, APTES) whose amine groups were then available for the attachment rhodamine-B molecules (RdB). After surface modification with APTES, prepared white powder was dispersed in deionized water, and RdB/EtOH solution was then added and the mixture was stirred vigorously at room temperature for 12 h. RdB labeled LDH nanoparticles (ZN–RdB) were then collected by centrifugation, washed with water/EtOH, and then freeze-dried. To label the RH molecules with Rdb, first the drug (RH) was dispersed in 10 ml of deionized water. A predetermined amount of RdB in water was the added to those dispersion drop wise. The reactions were carried out for overnight at room temperature with vigorous stirring in dark. After completion of the reaction, RdB labeled RHs were thoroughly washed with ethanol and deionized water. Being a hydrophobic fluorescent dye, the drug molecules were efficiently labeled with rhodamine-B. RdB labeled RHs are abbreviated as RH–RdB. To get PCL nanohybrids of ZN–RdB, ACS Paragon Plus Environment

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first ZN–RdB was dispersed in DCM and followed by its addition to PCL solution in DCM (25% ZN–RdB w/w with respect to PCL) and then the whole solution was stirred for 5 h to ensure proper mixing. The mixture solution was kept for 24 h in a fume hood for solvent evaporation and then vacuum dried at room temperature for an additional 24 h. This is designated as PN‒RdB. The uptake of hybrid nanoparticles by HeLa cells was observed using inverted fluorescence microscopy (DMILLED DFC3000G, Leica, Germany) and quantified by fluorescent intensity measurement through a microplate reader. In brief, 2 × 104 HeLa cells were seeded in 24 well plate for 24 h with 500 µl DMEM consisting of 10% FBS and 100µg ml-1 Penicillin-Streptomycin. The suspension of the rhodamine B labeled materials and pure rhodamine B were treated to the cell and incubated for 1 h, 3 h, 6 h, 12h, 24 h, 36 h and 48 h to understand the effect of incubation time to cellular uptake. After incubation, extracellular materials were removed by washing the cells with PBS and then cells were lysed in PBS containing 0.2% Triton X-100, then incubated further for another 15 minutes. The plate was centrifuged to remove cell debris and transfer the supernatant to 96 well plates to measure the fluorescence from rhodamine B at λEx = 540 nm and λEm = 625 nm. For fluorescence imaging cells were incubated for 1, 6, and 24 h and then rinsed three times with PBS buffer followed by fixation of the cells with 4% paraformaldehyde for 15 min at room temperature. Fluorescence images of cells with rhodamine B labeled nanohybrids and pure rhodamine B were taken using inverted fluorescence microscopy. Biodegradation: The biodegradability of PCL and its nanohybrid at ambient temperature has been performed in enzyme media by measuring weight loss at predetermined time. In vivo drug release study: 8-10 weeks old Charles Foster albino female rats, weighing 130±20 g, were purchased from the central animal house of the Institute of Medical Sciences, Banaras Hindu University, India. They were provided a standard laboratory diet and water. The study protocol was ACS Paragon Plus Environment


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approved by the animal ethics committee of Banaras Hindu University (Letter No: Dean/2016/CAEC/193) and all the animal studies were carried out in accordance with the approved guidelines and regulations. Pure RH, ZN‒R, ZP‒R and PNR formulations were injected intraperitoneally at the dose of 10 mg drug / Kg body weight and equivalent amount in drug intercalated LDHs, and consisted of four rats per group. Blood samples (~ 0.4 ml) were collected from the supra orbital vein of eye into heparinized microfuge tubes at a time interval of 0.25, 0.50, 1, 2, 4, 8, 16, 24, 30, 48 and 72 h post-dosing. The plasma samples were collected after centrifugation of the collected blood samples at 5,000 rpm for 15 min and stored at –70 ± 10 °C until assayed. Concentration of released RH in plasma was estimated according to the literature reported method.30 The observed maximum plasma concentration (Cmax) and the time to reach the maximum plasma concentration (tmax) were obtained from the observed concentration versus time profiles. Liver and renal function test: For biochemical analysis of liver function test (LFT) and renal function test (RFT) mice in different groups were sacrificed on the 4th day of CDDP treatment. Blood was collected directly from the heart and serum was separated, stored at –70 ± 10 °C until the analysis could be completed. Biochemical analyses included measurement of the activities of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) along with urea and creatinine level. Histopathological evaluation: At the end of in vivo experiment, rats were sacrificed and major organs, such as liver, kidney, spleen, etc., were separated and fixed using 10% buffered formalin solution. The tissues were then cut into sections ~ 5 µm thickness using a microtome and were stained with hematoxylin and eosin (H&E) for histological investigations. Histopathology of the


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tissues at the injected site has been performed to understand the local toxicity effect caused by material/drug embedded material (ZN-R / PN-R).

Statistical analysis: Results are presented as the mean value ± standard deviation (SD). Statistical analysis is made using analysis of variance (one-way ANOVA with t-test).

Results and Discussion: Synthesis of 2-D layered double hydroxide nanoparticles: Layered double hydroxides have been synthesized from the zinc and iron salt with three different interlayer anions of nitrate (NO3-1), carbonate (CO3-2) and phosphate (PO4-3) with varying charge density. Elemental composition and the presence of different anions are confirmed through Energy Dispersive X-ray Spectrometry (EDS) showing characteristic emission peak of Fe and Zn in addition to separate nitrogen and phosphorous peak in ZN and ZP, respectively, (Fig. 1a) and the detailed composition of all three LDHs are mentioned in supplementary Figure S1. The shape and size of the LDHs particles are found to be circular platelet-like with lateral dimension of ~80 ±4 nm with moderate distribution of particle size as examined through bright field TEM images (Fig. 1b). The crystalline natures of the LDH nanoparticles are shown by presence of concentric diffraction rings, characteristics of Zn based LDH; (018), (110) and (113) planes, as observed through selected area electron diffraction (SAED) patterns of ZN and ZP nanoparticles (Fig. 1c).31,32 The disc like morphology of the LDH is also confirmed through AFM images (Fig. 1d) with average particle diameter of ~85 ±4 nm, similar to the dimension observed in TEM images. Slight agglomerated surface morphology has been noticed in SEM images with the particle dimension of 91 ±7 nm (Figure 1e). This is to mention that better dispersed morphology has been observed in TEM from its sample preparation using dilute concentration and considered to be real dimension of the LDH ACS Paragon Plus Environment


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nanoparticle. The shape, size and composition of the other LDH have been presented in supplementary Figure S3. However, various charge containing counter anions (NO3-1, CO3-2 and PO4-3) in LDHs have been synthesized for the intercalation of biologically active species through ion exchange reaction for therapeutic application from a sand witched / hidden state.

Intercalation of drug within 2-D LDH and its embedment in polymer: Negatively charge drug (raloxifene) has been introduced within LDH galleries through ion exchange reaction by replacing the NO3-1, CO3-2 and PO4-3 ions from the respective LDHs. The intercalation of drug is understood from the appearance of a new peak at 2θ ∼ 6.7o in ZN-R (indicated with the ‘∗’ mark), corresponding to an increase in the interlayer distance of 1.32 nm from the strong peak of pure ZN at 2θ ~ 11.34o (d ~ 0.834 nm) assigned for (003) plane of pristine LDH (Fig. 2a). The intercalated peak becomes less intense in PN-R (polymer nanohybrid) due to disordered structure of ZN-R in polymer matrix. The higher order diffraction peaks, (006) and (009) plane, along with other crystallographic planes of (110) and (113) indicate the crystallized hydrotalcite-like structure with a rhombohedral packing of the 2-D LDHs.33,34 However, the crystalline peaks become weaker and broader in ZN-R and PN-R (nanohybrid) due to incorporation of drug within the gallery in ZN-R and its dispersed phase structure in PCL matrix as compared to pure ZN. Pristine PCL shows sharp crystalline peaks at 21.5o and 23.85o corresponding to the (110) and (200) crystallographic planes which have slightly shifted after the embedment of ZN-R in PCL matrix presumably due to interaction. Further, the effect of drug intercalation has a marked effect on the morphology of ZN-R where the stacking of platelets is clearly visible with lateral dimension of 90±7 nm (Fig. 2b) due to agglomeration caused by drug molecules. The bright field TEM images of ZC-R and ZP-R also exhibit similar characteristics with average lateral dimension of 88 ±2 and 91 ±3 nm, respectively (supplementary Figure S4). The SAED pattern of ZN‒R particle is shown ACS Paragon Plus Environment

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by the presence of concentric diffraction rings (inset of Fig. 2b) against a corona around the pointer in PN-R, implying the existence of large poly-crystalline structure of LDH in polymer matrix. On the other hand, dispersed morphology of ZN-R is evident in PN-R. Moreover, discrete particle morphology is noticed in AFM micrograph with periodic height profile of ZN-R against smooth surface morphology in pure PCL which subsequently periodic again in PN-R (nanohybrid) showing the marked effect of drug embedded nanoparticle in PCL matrix (Fig. 3c). The size distribution of LDH particle before and after drug intercalation has also been shown through dynamic light scattering (DLS) measurement of ZN, ZN-R, PCL-RH and PN-R which were found to be 100±4, 110±3, 315±4 and 332±3 nm with polydispersity index (PDI) of 0.19, 0.22, 0.17 and 0.20 respectively (supplementary Figure S5). Surface charge, a key parameter for biologically active molecule, has been measured through zeta potential and high positive and negative values of 48 ±5 and ‒33 ±4 mV are detected for ZN (pure LDH) and RH (pure drug), respectively, and drug intercalation in LDH reduce its zeta potential to 27 ±3 mV. Further, the zeta potential of ZN-R embedded nanohybrid shows a moderate value of ‒18 ±2 mV in PN-R considering the zeta potential of pure PCL of ‒22 ±2 mV (Fig. 2d). The zeta potential values of ZC-R and ZP-R systems have been found to be 31 ±3 and 28 ±2 mV, respectively. However, surface charge has been regulated by intercalating the drug into LDH layers and subsequently embedding it into the polymer matrix for improved drug release in biological media.

In vitro controlled release of drug: Sustained release of drug is essential especially for cancer treatment to keep the concentration of drug within the therapeutic window for longer period of time to avoid disastrous side effect of the drug. In vitro release profile of raloxifene from the three different drug intercalated LDHs and PCL nanohybrid is investigated through absorption spectroscopy ACS Paragon Plus Environment


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considering the peak position at 285 nm. Figure 3a illustrates the cumulative percentage release of raloxifene in PBS media (pH ~ 7.4) at 37 oC. ZP‒R (phosphate ion as intercalant in LDH) exhibits burst release kinetics, about 50% of the drug is released in just 15 min and nearly 100% release occurs in 5 h. Though initial fast release is observed for ZN-R (nitrate ion as intercalant in LDH) while it follow a slow release afterwards performing a biphasic elution profile and continue to release for 36 h. Hence, there is gradual controlled release of drug from ZP-R to ZC-R to ZN-R with varying charge density of intercalant from 3 (phosphate) to 1 (nitrate) presumably due to greater interaction between drug and LDHs. When the drug is embedded directly in PCL (PCL‒RH), it showed a steady release while unique sustained release of drug is obtained only from PN-R where drug intercalated LDH is embedded in polymer. Interestingly, the initial release rate of drug is sufficiently high (35% in 24 h), as per the requirement of therapeutics, followed by a more sustained release for longer period of time (60% in 80 h). However, a steady but sustained release is observed from PN-R (polymer nanohybrid) in longer period of time. In case of drug intercalated LDHs, release of the drug molecules from these intercalates took place through ion-exchange of the drug anions by phosphate ions present in PBS medium. On contrary, the entrapped drug molecules are released from the intercalated LDH to polymer matrix first followed by its release to actual media through diffusion process in case of PN-R (polymer nanohybrid). The typical drug release mechanism has been shown in Fig. 3b showing first release for ZN-R (direct release) against significantly sustained release in PN-R (via polymeric media) through diffusion process. This is to mention that water/PBS media permeation has to occur first within the polymeric bulk when diffusion of drug can only be a feasible process in PN-R (polymer nanohybrid) and delayed diffusion or sustained release in PN-R is explained from the three step process; i) permeation of solvent in to polymer matrix, ii) diffusion of drug molecule from intercalate to polymeric media, and iii) diffusion of drug from polymeric substrate to actual release media (liquid). The


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experimental evidence of considerably slow drug release from polymer matrix has been given in Fig. 3a while very low permeation of water molecule in PCL matrix is well known.35 Thus, the (i) and (iii) processes are quite slow and are responsible for sustained release in PN-R against the only (ii) process is operative in ZN-R or other drug intercalated LDHs make them fast release systems being the noticeably fast diffusion process as also evident from the fast drug release kinetics in Fig. 3a. The release rate of the drug molecules from the intercalates is mainly governed by the factors like drug-host interactions, nature of the anions present in the interlayer region and the anions present in the PBS and LDH host layer and will be discussed in next section. However, it is evident that the release of drug molecules from the PN‒R nanohybrid is more prolonged and sustained as compared to only drug intercalated LDHs systems. To understand the drug release kinetics, a number of kinetic models such as Zero-order, first-order, Higuchi, Korsmeyer–Peppas, Elovich equation, parabolic diffusion, and modifiedFreundlich kinetic models have been exploited to all these systems.36,37 The linear correlation coefficient (r2) and other fitting parameter values obtained from the linear fittings of the drug release data with various mathematical models are given in supplementary Table S1. Among these models, zero-order, first-order and Higuchi models result poor r2 values ranging from 0.679 to 0.912 and are found not suitable to explain release mechanism (supplementary Figure S6) while it is found that modified-Freundlich and Korsmeyer–Peppas models are most satisfactory for describing the mechanism of RH release from drug-LDH intercalate and PCL-(LDH-drug) nanohybrid, respectively (Fig. 3c). The modified Freundlich model describes heterogeneous diffusion from the flat surfaces via both ion exchange and diffusion controlled phenomena. ZN‒R, ZC‒R and ZP‒R systems are best fitted with modified Freundlich model with high linear correlation coefficients (r2 ~ 0.98–0.99). For PCL-RH and PN‒R systems, Korsmeyer–Peppas model explains the release phenomena more reasonably (r2 = 0.992 and 0.991) leading to the ACS Paragon Plus Environment


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exponent ‘n’ value of 0.47 and 0.51, respectively, indicating the non-Fickian nature (n ≥ 0.45) of drug release from the hybrid following the transport case-II. The delayed diffusion of the RH molecules from PCL–RH system is presumably due to the presence of crystalline fringes present in the PCL matrix, which create a maze, called a ‘tortuous path’.23 However, the mechanism presented in the schematic demonstrates fast and slow release of drug from LDH-drug intercalate and polymer-(LDH-drug) nanohybrid.

Understanding the controlled release from interactions: The nature of interactions between the drug and host molecules (LDH/PCL) is crucial to understand the differential controlled release behavior of the drug molecules from various drug vehicles. Figure 4a shows the XPS spectrum of Fe 2p (originated from LDH host layers) before and after drug intercalation exhibiting two prominent signals at 711-715 eV and 725-727 eV range corresponding to Fe 2p3/2 and Fe 2p1/2, respectively, along with a satellite peak appeared at around 719 eV, attributed to Fe3+ states.38 The binding energies (BE) of both Fe 2p3/2 and Fe 2p1/2 states have shifted to higher energy in drug intercalated LDHs. The BE of Fe 2p3/2 peak of ZP-R has shifted 0.06 eV (712.51 → 712.57 eV) as compared to pure ZP while 0.24 eV (725.78 → 726.02 eV) peak shift occurs for Fe 2p1/2 state. The corresponding peak shifts in ZN-R are 0.53 (711.18 → 711.71 eV) and 0.60 eV (725.16 → 725.76 eV) for Fe 2p3/2 and 2p1/2 state, respectively, clearly indicating greater interaction between drug and LDH in ZN-R vis-à-vis ZP-R as evident from greater peak shifting of binding energy. Similar shifting of BE of Fe 2p peaks also occurs for ZC-R and lies in between ZP-R and ZN-R suggesting better interaction than ZP-R but lesser interaction than ZN-R. Further, Zn 2p3/2 peaks shift 0.03, 0.1 and 0.52 eV for ZP‒R, ZC‒R and ZN‒R systems, respectively, as compared to their respective pure LDHs i.e. ZP, ZC and ZN. Similarly, peak shifting for Zn 2p1/2 state are 0.05, 0.22 and 0.54 eV for ZP‒R, ZC‒R and ZN‒R, respectively, visACS Paragon Plus Environment

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à-vis their respective pure LDHs. The greater shifting of BE from ZN and ZN‒R is attributed to better drug‒LDH interaction. Hence, XPS measurements clearly indicate that drug molecules strongly interact with ZN while the extent of interactions goes down in ZC and ZP gradually which is responsible for the trend of drug release discussed in previous section. Drug intercalated LDH, ZN‒R exhibits absorption peaks at 228 and 395 nm having prominent red shifting as compared to absorption peak of pure LDH (ZN) at 220 nm due to the presence of nitrate anions in the interlayer galleries (Fig. 4b).39 The peak at 364 nm of pure drug (Raloxifene) has shifted to 395 nm in ZN-R confirming greater interaction between RH and ZN against meager shifting in ZP‒R with the absorption peak at 222 and 391 nm suggesting relatively poor interaction between drug and ZP (LDH) (supplementary Figure S7). Further, red shifting is noticed in polymer nanohybrid (PN-R) but the overall high absorption restricts to locate the specific absorption peaks. FTIR spectra of pristine ZN exhibits a peak at 3468 cm−1, attributed to the stretching vibration of O‒H groups both in the brucite-like layers of LDH and from the interlayer water molecules, which has shifted significantly to 3435 and 3430 cm−1 in ZN-R and PN-R, respectively, indicating strong interaction between drug and ZN which further enhances when embedded in polymer matrix in PN-R (Fig. 4c). Moreover, a prominent drug peak at 1597 cm−1 due to –C–O–C– stretching frequency has shifted to 1595 and 1592 cm−1 in ZN-R and PN-R, respectively, also indicate relative higher interaction when drug embedded ZN is embedded in polymer matrix. Other distinct peaks of pure ZN at 1382 cm−1, assigned to stretching vibration of the NO3− groups, 1728 (ν(C=O)) and 1172 cm−1 (ν(C–O–C)) peak of pure PCL appear for PN-R suggesting the presence of the components in polymer nanohybrid.23,40 The absorption bands below 1000 cm−1 are due to M‒O and M‒O‒H vibration modes of LDH layers and the patterns are very similar to previous literature.10,40 The relative shifting in ZP-R as comparison to pure ZP is quite



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low indicating less interaction in ZP-R vis-à-vis ZN-R and is presented in supplementary Figure S8. Differential scanning calorimetry (DSC) was employed to understand the interaction between drug molecules and different types of LDH as host layers and subsequently their embedment in polymer matrix both qualitatively and quantitatively. The depression of melting temperature along with heat of fusion is considered to be the extent of interaction between the components. The melting point of intercalated drug in ZN-R has lowered by 41 oC to 223 °C from the melting point of pure drug at 264 °C (Fig. 4d) while this temperature decrease becomes 13 oC only (264 → 251 °C) in ZP-R (supplementary Figure S9) indicating greater interaction in ZN-R system as compared to ZP-R. Further, the heat of fusion of ZN-R has reduced significantly to 68 J.g-1 from the pure drug of 85 J.g-1 also supports the greater reduction in ZN-R vis-à-vis ZP-R (73 J.g-1) due to better interaction in ZN-R. This is to mention that the melting of PCL is affected by the presence of ZN-R and increases the melting point of PCL to 64.4 oC from the pure PCL melting of 59 oC with significant peak broadening (inset of Fig. 4d). The effect of drug melting is not clear as the PN-R degrade before the melting temperature of drug. Thermogravimetric analysis (TGA) is carried out to measure the thermal stability of the samples under nitrogen atmosphere through weight loss under heating program. Pure ZN exhibits weight loss at 150 °C due to loss of adsorbed and intercalant water from LDH galleries while this weight loss temperature has increased to 218 and 237 °C for ZN-R and PN-R, respectively, indicating better thermal stability after drug intercalation and subsequent embedment in polymer matrix. This is worth mentioning that the degradation of pure drug and PCL are found to be 305 and 355 °C, respectively (supplementary Figure S10). However, the sand witched structure where and polymer wrapping of the drug embedded LDH enhances the thermal stability for high temperature uses. Thus, the spectroscopic and thermal measurements clearly indicate that the interaction between drug and LDH increases


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from phosphate to carbonate to nitrate as intercalant in the order of ZP-R < ZC-R < ZN-R and significant interaction occurs in PN-R between ZN-R and PCL matrix. The origin of interaction between matrix and drug is predominantly dipolar in nature. This is noteworthy to mention that greater interaction with drug and matrix is reflected in their drug release profile and sustained release of drug in PN-R (polymer nanohybrid) is greatly envisaged from these relative grades of interaction with drug and matrix (either LDHs or polymer).

Effect of drug intercalation and embedment on cytotoxicity: Understanding the sustained release of drug from polymer nanohybrid after intercalating the drug molecules within the layers of LDH, now it is pertinent to study the effect of sustained release on cellular uptake and thereafter cell health. HeLa cell line (cancer cell) is chosen to evaluate the performance of anti cancer drug like raloxifene from various hosts having regulated drug release rate keeping the amount of drug same. Cell viability is assessed through MTT assay after incubating the cells for 24, 48 and 72 h in different concentration of raloxifene (10, 30 and 60 µg/ml) in pure state and equivalent amount in drug loaded LDHs and PCL nanohybrid demonstrating the significant cytotoxicity of PN-R (polymer nanohybrid) towards HeLa cell as compared to pure Raloxifene (Fig. 5a). In other words, killing efficiency of PN-R is considerably high (rapid and consistent decrease of cell density with time) as compared to drug intercalated LDHs (less decrease of cell density vis-à-vis PN-R) and pure drug (only meager decrement in 3 days). Further, ZN-R exhibits better performance in long run as compared to ZP-R even though the initial efficiency of ZP-R is slightly high because of faster drug release kinetics as evident from Fig. 3a. ZC-R exhibits cell killing efficacy in between ZN-R and ZP-R (supplementary Figure S11) in commensurate with the in vitro drug release profiles. The cell viability is supported by the optical images of cell after the treatment of HeLa cell with the above mentioned drug/drug release systems showing



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minimum cell density in PN-R treated system against the large number of live cells still exist after 3 days of treatment with pure drug (Fig. 5b) and follow the similar trend of cell viability measurement observed in Fig. 5a. The IC50 (half maximal inhibitory concentration) is a measure of the effectiveness of a drug. The IC50 of free RH, ZN-R and PN-R against HeLa cells are measured to be 0.79, 0.58 and 0.52 nM, respectively. This is worthy to mention that direct embedment of drug in PCL (PCL-RH) has very low impact on the killing efficiency of HeLa cells as observed from cell images (high cell density) and cell viability study (higher viability) which shed light on the importance of intercalation of drug within LDH followed by its embedment in PC matrix for greater efficiency of drug release and killing effectiveness of PN-R system as compared to others. This finding may be understood from the better bioavailability of the drug in drug intercalated LDHs and PN−R as compared to PCL-RH or free drug which will be uncovered in details in the next section. However, above mentioned different drug concentrations are chosen randomly to undertake the trial experiment for estimation of the efficacy of the drug loaded showing better killing efficiency at higher dose (supplementary Figure S12). The cell viability (survival rate) after treatment of 10 µg/ml of pure RH or equivalent amount of drug loaded in different materials was measured to be 81 ±1.3, 78 ±1.7, 61 ±1.2, 58 ±2.4 and 46 ±2.1% for RH, PCL−RH, ZP−R, ZN−R and PN−R, respectively. After treatment with 30 µg/ml of pure RH or equivalent amount of drug loaded materials, the cell viabilities decrease to 67 ±1.4, 58 ±1.1, 43 ±2.1, 38 ±1.5 and 26 ±2.1%, respectively, while 45 ±1.3, 37 ±1.5, 19 ±1.7, 20 ±1.9 and 18 ±1.1% cell viabilities were obtained after treating with 60 µg/ml of pure RH or equivalent amount of drug loaded in various materials. This is to mention that pure materials e.g. ZN, ZC, ZP and PCL are excellent biocompatible where cells can grow nicely on top of these pure materials without drug loading and thereby found to be good drug delivery vehicles (supplementary Figure S13) which in turn confirm that drug release


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from the drug intercalated systems is responsible for killing of the cells. Again, cell viability of the developed materials also has been performed using fibroblast (NIH-3T3) cells to investigate whether there is any toxicity introduced by these materials to normal cells. Percentage of cell viabilities has been observed to be increased with increasing incubation time for all the three samples confirming their biocompatibility (supplementary Figure S14a). The biocompatibility of the samples has also been further confirmed through fluorescence imaging after staining the cells with acridine orange and ethidium bromide (supplementary Figure S14b). Consistent with the MTT assay measurement, Figure S14b also confirms the presence of healthy cells. These results therefore clearly suggest that the PCL, LDH and PN possess excellent cytocompatibility. These results clearly demonstrate that PN−R exhibits the highest in vitro anti-cancer efficiency (cell mortality) in comparison to pure drug and drug loaded LDHs. Superior in vitro anti-cancer activity has been reported for sustained release systems using MMT41 and superparamagnetic iron oxide42 in drug loaded polymer matrix (PLA and PLGA) as compared to the pure drug. To elucidate the mechanism of cell death and to confirm the potential of nanoparticles in inducing apoptosis, cells are stained with acridine orange (AO) and ethidium bromide (EtBr) and are examined using a fluorescent microscope. This double staining helps us to distinguish the normal cells from apoptotic cells based on the permeability of cell membrane. Cell membrane being permeable, AO binds with DNA exhibiting green fluorescence while EtBr is selectively taken up by apoptotic cells and stains the condensed nuclei with red fluorescence.43 AO/EtBr staining cells are observed as yellow-orange colored cells which are apoptotic and necrotic cells provide red color fluorescence due to their loss of membrane integrity while healthy live cells are observed as green color fluorescence.10,43 In vitro antitumor activity of raloxifene loaded materials is monitored using HeLa cells for different incubation time showing green region with yellowish nuclear fragments in ZN−R and PN−R treated cells due to apoptosis against the green colored cells (live) using PCL-RH and pure drug like ACS Paragon Plus Environment


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control system (Fig. 5b). Therefore, cell viability and cell morphology studies strongly suggest that PN−R has significantly better in vitro anticancer efficacy as compared to pure drug or PCL-RH at the same drug concentration and exposure time. Hence, the polymer nanohybrid (PN−R) enhances the therapeutic effect using similar quantity of drug used directly or intercalated within LDHs.

Nanoparticle induced cell adhesion: The cell adhesion is an important process to evaluate a material for its biocompatibility and the cells need to be adhered first for its proliferation on any substrate. LDH pellets, pure PCL and its nanohybrid films are evaluated by qualitative observation of morphological features as well as cell viability measurements which are adhered onto the substrate. LDH embedded PCL (without drug) exhibits highest cell adhesion as compared to pure PCL and ZN (Fig. 5c) showing better biocompatibility of nanohybrid vis-à-vis pure PCL and LDHs. Interestingly, the cell morphology on polymer nanohybrid (PN) without drug has the flat, well-spread and elongated morphology with better cell to cell contacts against the squeezed morphology with reduced cell-cell contacts on pure PCL and ZN strongly indicate the polymer nanohybrid (without drug) is undoubtedly a better biomaterial (Fig. 5d). This is to mention that the cells are nicely adhered and spread moderately on tissue culture polystyrene plate surface (control). However, better biocompatible nature of polymer nanohybrid (PN without drug) has been explained from its greater surface wet ability as obvious from the contact angle measurement of 67o and 95o for PCL-LDH (nanohybrid) and pure PCL, respectively (Supplementary Figure S15). The greater hydrophilicity (low contact angle) is more adaptive for the cells to grow on its surface and the lowering of contact angle in nanohybrid is explained from the very low contact angle of pure LDH (~ 53o), whose contribution is ~25% in the nanohybrid. Higher water contact angle of PCL makes it hydrophobic and as a consequence, it interacts with the cells inadequately, leading to weak adhesion. However, because of hydrophilic ACS Paragon Plus Environment

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character of the nanohybid (low contact angle), it interacts with the cells strongly leading to greater cell adhesion. Thus, an increment in hydrophilicity of PCL-LDH films facilitates cell adhesion and spreading on its surface which has strong influence on the killing efficiency for the measurement of cell proliferation in drug loaded specimens as observed in Fig. 5a. Further, the cell adhesion is also influenced by the surface roughness of the film and greater surface roughness of nanohybrid film, as evident from the AFM height profile measurement in Fig. 2c, as compared to pure PCL help improving the cell adhesion.44 The cell adhesion study using a normal cell (fibroblast, NIH-3T3) shows significant different results than that of tumor cells (HeLa). Both pure LDH and PN (polymer nanohybrid) show enhanced cell adhesion behavior towards cancer cells as compared to fibroblast cell while pure PCL weakly adheres on both types of cells (supplementary Figure S16). This cell specific adhesion of LDH and PN helps to target cancer cells in comparison to the normal cells (fibroblast) and thereby reducing the adverse side effects towards the normal tissues.

Controlled cellular uptake: The permeation of biologically active molecule from the surrounding to inside cell cytoplasm through cell membrane is necessary to understand the effect of biologically active molecules towards cell. Here, drug molecules from the drug delivery vehicle are in contact with the cell and its efficacy to kill the cells mainly depend on its uptake inside the cell through cell membrane. In this work, drug is intercalated within LDH and subsequently it is embedded in PCL matrix. If LDH stacks penetrate through cell membrane, drug can easily be delivered into cell cytoplasm and can be compared with the permeation of pure drug in a similar manner. To locate the LDH and drug molecules inside the cell, drug (RH) and LDH nanoparticles are labeled with fluorescence rhodamine B (RdB) to validate the efficacy of the carrier to transport specific biologically active molecules into the cells. For first couple of hours, pure drug labeled with RdB



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couldn’t penetrate the cell membrane, as evident from no fluorescence up to 5 h while certain amount of drug has been penetrated into the cell in 24 h (Fig. 5e). In contrast, sufficient fluorescence has been noticed for polymer nanohybrid (PN-RdB; Rhodamine B tagged ZN and subsequent embedment in PCL) in less than 1 h predominantly in the cell wall in the form of stacks and gradually entered the cell cytoplasm in uniform and dense manner as clear from the fluorescence image while meager flurorescence is observed in PCL-RdB, where rhodamine B tagged drug was embedded directly in PCL in 24 h. Rhodamine B tagged ZN is also entering into cell cytoplasm but significantly less intense than PN-RdB system suggesting greater cellular uptake using polymer nanohybrid vis-à-vis LDH or PCL. The quantitative measurement of cellular uptake has been shown through mean fluorescence intensity using microplate analyzer in Fig. 5f showing rapid initial uptake up to 6 h incubation for RdB tagged LDH. Even though the initial fluorescence is low in polymer nanohybrid, presumably due to initial slower release rate, it rapidly increases after 12 h of incubation and exhibit higher fluorescence than RdB tagged LDH. Further, the uptake using PCL-RdB (RdB tagged drug is directly embedded in PCL) is quite low as compared to LDH systems, either pure or embedded in PCL. However, PN–RdB (nanohybrid) exhibits gradual and significant uptake up to 48 h of incubation against lesser uptake using pure LDH and PCL leading to differential total uptake of biological molecules (RdB) into cell using pure RH-RdB, PCL-RdB and PN-RdH as shown in a cartoon in Fig. 5g. Hence, it is concluded that free drug molecules (RH) cannot enter into the cell in sufficient quantity primarily due to its negative surface charge facing repulsion from the negatively charged cell membrane while the positively charged LDH nanoparticles get attracted to negatively charged cell membrane facilitating the cellular uptake using LDHs through endocytosis mechanism.12,45 Choy et al. demonstrated the clathrin-mediated endocytosis, mainly responsible for the efficient uptake of LDH nanoparticles with an upper limit of the lateral dimension of 200 nm and in this study the LDH size of ~80 nm helps improving the


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cellular uptake to a great extent.46 The enhancement of cellular uptake of LDH nanoparticles depends on the factors like concentration and time of incubation45-47 and the significant enhancement of fluorescence intensity using nanohybrid over PCL may be understood from the fact that with incorporation of LDH in nanohybrid enhances the cellular adhesion properties which facilitates better interaction between the cells and materials through docking phenomena.48,49 Now, it is pertinent to understand the greater cellular uptake in nanohybrid as compared to pure polymer or LDH. Cellular uptake is a two step process, starts with an initial adhesion of the material to the cell and interactions with the lipids, proteins and other components of the cell membrane. This process is followed by the activation of an energy-dependent uptake mechanism.50 Material with greater cellular adhesion exhibits a concomitant increase in uptake efficiency. Here, nanohybrid exhibits best cell adhesion over its surface mainly because of its better hydrophilic behavior (lowest contact angle) and interaction where cells can completely spread on the surface and subsequently it helps greater cellular uptake through insertion of drug intercalated LDH within the cytoplasm through endocytosis process and kill the cancerous cell in a best possible manner as shown in the carton (Scheme I) while squeezed cell morphology on pure PCL surface (presumably due to hydrophobic behavior) cannot facilitate much for its insertion into cell through endocytosis following less cellular uptake and poor efficacy to kill the cancerous cells. Initially, the positively charged rhodamine–B labeled LDH docks on the negatively charged cell membrane and can easily go inside the cells through endocytosis

12,45

whereas the negatively charged pure drug molecules

follow slow diffusion through the negatively charged cell membrane. Although, ZN-R and PN-R carry opposite charge, they exhibit similar trend in cellular uptake performances. From quantitative analysis of cellular uptake of ZN-RdB and PN-RdB as a function of concentration, it is found that cellular uptake performances have increased with the concentration of ZN-RdB from 10 - 50 µg/mL. However, cellular uptake efficiency has been found to decrease significantly with further



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increase in the concentration from 50 - 500 µg/mL (supplementary information Figure S17). LDH has a strong tendency to form agglomerate in culture media. On contrary, no considerable lowering of cellular uptake is observed using PN-RdB even at higher concentration. Hence, well dispersive nature and strong cellular adhesion of PN-R nanohybrid are responsible for its enhanced cellular uptake performances.

Effect of controlled drug release in animal model: Understanding the controlled release of drug in cell line and its efficacy to kill the cells differentially, it is important to know how effective they are in actual animal model. The goal of this study is to investigate whether these carriers can successfully deliver and control the drug (RH) concentration in blood plasma within therapeutic range over a prolonged time period and thereby to reduce the adverse side effects of anticancer drugs (RH) from its burst release. To compare in vivo performance of pure RH and RH intercalated in LDH and subsequent embedment in polymeric material, the concentration of drug in the blood stream is measured after an intravenous dosage of 10 mg.kg−1 of RH, either pure drug or equivalent amount in various drug vehicles, in female Charles Foster albino rats. The mean plasma drug concentration is plotted in Fig. 6a as a function of time. The minimum concentration of raloxifene needed to detect a significant decrease in cell number is 10−9 M (i.e. ~ 0.5 ng ml−1) for human prostate cancer cell lines51 and, therefore, a threshold of 1 ng ml−1 RH is chosen for this purpose. A rapid declining profile of RH concentration in plasma for injected pure drug has been observed with an initial burst release (Cmax ~321 ng / ml at 45 min) and complete release of drug occurs within only 8 h after administration. On the other hand, drug intercalated in LDHs exhibit considerable sustained release up to 30 and 40 h for ZP‒R and ZN‒R systems, respectively, with minimal initial burst release (Cmax ~120 and 112 ng/ml, respectively). Interestingly, the sustained release in the blood stream is continued up to 100 h (more


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than 4 days) for polymer nanohybrid (PN-R) system with drastically suppressed burst release, only up to ~45 ng/ml and, thereby, confirms the greatest efficacy of the polymer nanohybrid as the drug carrier over the drug intercalated LDH and pure drug. Thus, the animal model demonstrates the effectiveness of the polymer nanohybrid as the superior and versatile drug delivery vehicle for cancer treatment and found to be much better effect than injecting pure drug for cancer treatment. It is important to note that polymer nanohybrid delivers drug for more than 4 days (>100 h) continuously in the blood stream against the very short existence of drug (10 h) in the blood plasma when same amount of pure drug is injected directly.

Monitoring the effects of controlled release in other organs: Sustained drug release in blood stream has been noticed in animal model but the effect of this new drug delivery vehicle towards other sensitive body parts/organs should be understood for its real application. Liver function tests (LFT) have been employed to examine hepatic dysfunctions caused by drug administration. Activity of liver enzymes such as ALT and AST have been studied to assess hepatotoxicity in CF albino female rats after intravenous administration at 10 mg drug / Kg body weight and equivalent amount of drug in other drug carriers. Significant increase in ALT (~115%) and AST (~175 %) activities have been found, in the rats treated with pure drug (RH), as compared to the control group values of ALT of 32 U/L and AST of 115 U/L after 24 h while meager increase is observed in the rats, treated with polymer nanohybrid, of ALT (∼9%) and AST (∼10%) (Fig. 6 b & c). However, marginal increment of ALT and AST is observed in rats treated with polymer nanohybrid against huge increase in pure drug treated rats indicate that the lever is working satisfactorily using polymer nanohybrid as the drug delivery vehicle. This is to mention that ALT and AST increment is slightly more for drug intercalated LDHs as compared to PN-R and higher value of ZP-R is explained from the burst release in comparison to ZN-R. However, a



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gradual abatement of liver enzymes activity with time is observed for all the rats but the absolute value is considerably high in case pure drug treated rats even after 3 weeks of administration. Serum urea and creatinine levels are also studied to further monitor the renal toxicity of all the rats (control and treated groups). For first few days after the treatment, no significant increase in serum urea and creatinine levels is observed (supplementary Figure S18 a & b). However, rats treated with pure RH exhibit significant elevation in serum urea and creatinine levels. The increase in serum urea and creatinine level is about ∼114 and ∼123%, respectively, after 7 days of administration (Fig. 6d) with marginal increment in rats treated with polymer nanohybrid or drug intercalated LDHs. Further, the elevated levels of serum urea and creatinine have gone down considerably at longer period except for those rats treated with pure drug and fascinatingly PN‒R treated group show the depression levels of serum urea and creatinine up to 14 and 11 %, respectively. Our results also indicate that lever is badly affected by the pure drug just after its administration while kidney is affected slightly at a later stage. Importantly, these bad effects of pure drug have been avoided drastically through drug administration using polymer nanohybrid and, thereby, hepatic dysfunctions and renal toxicity are minimally affected with this novel drug delivery vehicle as opposed to the severe damage of lever and kidney in conventional treatment of cancer. Moreover, triglyceride level in blood has increased considerably in rats treated with pure drug after 7 days of administration to 80 mg/dl (10% increment) against meager changes in rats treated with polymer nanohybrid of 74 mg/dl (1.5% increment) and drug loaded LDHs to 75 mg/dl (3% increment) with the control value of 73 mg/dl (supplementary Figure S18c). Histopathological evidence of differential organ damage due to drug: Histopathological evaluations are commonly used to investigate organ-specific effects of chemotherapeutic drugs. In the present study, hematoxylin and eosin (H & E) staining of the main organs including liver, kidney and spleen are used to evaluate the toxic effects of pure drug and ACS Paragon Plus Environment

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drug intercalated/embedded materials. Analysis of liver tissues of rats in the control group shows normal architectural hepatocytes, which are large in size, hexagonal in shape with more or less centrally located one nuclei and homogenous cytoplasm (Fig. 6e). Similar results are observed for PN‒R treated rats indicating no damage of lever tissues even at longer period (21 days) after drug administration. In contrast, hepatotoxic symptoms such as portal triad having bile ductular proliferation, deformation in shape and sizes of the hepatocytes and some inflammatory reactions are clearly observed in rats treated with pure drug (indicated by blue arrows). Rats treated with ZN‒R and ZP‒R experience no obvious hepatotoxicity. However, no significant histopathological abnormalities or lesions are observed in kidney and spleen in all the groups (supplementary Figure S19). In conformity of the LFT results, damaged liver is obvious for pure drug treated rats against healthy lever in rats treated with polymer nanohybrid. It is worth mentioning that pristine LDHs have no toxic effects as illustrated by clinical chemistry and histopathology.10 This is to mention that there is no local toxicity at the injection site as confirmed from the histological evidence of the tissues at injection site (supplementary Figure S20). Significant progress in drug delivery is reported using polymer nanohybrid as novel drug carrier against pure drug administration which is currently being used for cancer treatment causing loss of appetite and nausea after chemotherapy. Drug is intercalated within the layered double hydroxide galleries through ion exchange reaction followed by its embedment in polymer matrix which release the drug slowly in the blood stream for more than 4 days continuously maintaining the therapeutic window limit as opposed to the toxic limit at lower release time followed by quick disappearance (< 10 h) from blood stream for pure drug. Faster release of drug causes toxicity to other organs like liver by crossing the higher window of therapeutic limit while sustained release using polymer nanohybrid retain the other organs healthy but kill the tumor cells effectively vis-àvis pure drug through its greater bioavailability by maintaining the hydrophilic hydrophobic design



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of the drug carrier. Importantly, the matrix polymer or its nanohybrid is green in nature as observed through their biodegradation behavior (supplementary Figure S21).

Conclusions In this study, a new anticancer drug delivery vehicle has been designed by intercalating drug within the layers of two-dimensional nanoplatelets of layered double hydroxide through ion exchange reaction followed by embedding them in a polymer matrix, called polymer nanohybrid. Elemental analysis of LDH and its shape, size and varying intercalants are verified using EDS, TEM, AFM and SEM studies. Raloxifene drug is intercalated within the LDH layers through ion exchange reactions. The level of drug intercalated LDH dispersion in polymer matrix is found to be uniform and surface charge (zeta potential) and roughness is modified in presence of LDH in polymer which is suitable for cell biology. There is considerable slow release of drug in LDH with decreasing charge density of intercalant from PO4-3 to CO3-2 to NO3− and significant sustained release is achieved from the polymer nanohybrid following two step release of drug from LDH to polymer matrix and then to release media considering very slow permeation of media into nanohybrid for greater tortuosity. The gradual increase of interaction between drug and LDH in three different LDHs (ZP-R < ZC-R < ZN-R) is measured using XPS, FTIR, UV-Vis and DSC studies and this increased interaction is reflected in their sustained drug delivery (better interaction leads to sustained release). Polymer nanohybrid (PN−R) exhibits the best in vitro anticancer efficacy as compared to pure drug or drug embedded polymer (PCL-RH) as evident from in vitro cytotoxicity experiment like cell viability. Cell adhesion in polymer nanohybrid is found to be superior even compared to pure polymer or pure LDH. Better bioavailability (cellular uptake) of polymer nanohybrid is demonstrated through tagging of fluorescence rhodamine B with pure drug and pure LDH and subsequent embedment in polymer. Better cell adhesion in polymer nanohybrid ensures


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greater cellular uptake which in turn improve the efficacy of cancer therapy. A model has been proposed for greater cellular uptake through better cell adhesion and thereby responsible for novel drug delivery vehicle using polymer nanohybrid. Sustained release of drug has been confirmed in vivo system using albino rats as a function of time showing healthy lever and other body parts using polymer nanohybrid against damaged lever using pure drug or drug intercalated LDH as revealed from the histopathological, lever and renal functional studies. Thus, a novel drug delivery vehicle has been developed having sustained release kinetics of drug without adverse side effects of anticancer drug.

ASSOCIATED CONTENT Supporting Information Supporting information is available free of charge on the ACS Publications website at http://pubs.acs.org. Figures S1-S21, Table S1.

Acknowledgements The author (SS) acknowledges the receipt of funding for his fellowship from the University Grants Commission (UGC), India. We gratefully acknowledge the financial support by Institute Research Project (Individual Faculty sanction No. IIT(BHU)/R&D/IRP/2015-16/2817) and SERB Project No. EMR/2015/001409. The authors are thankful to Central Instrument Facility Centre (CIFC), IIT (BHU), Varanasi, for SEM and AFM measurements.



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Figure 1: Elemental and morphological characterization of various LDH nanoparticles. (a) Energy dispersive X-ray spectrum showing strong signals of Zn and Fe, confirming the formation of ZN and ZP LDH nanoparticles; (b) High-resolution transmission electron microscopic image of platelet like shape with average size of ZN and ZP LDH nanoparticles of 81 and 83 nm, respectively; inset shows the histogram analysis of the particle size distribution; (c) SAED patterns of ZN and ZP LDHs showing various planes of LDHs; (d) AFM topographical images of the samples after spincoated on a SiO2/Si glass substrate; and (e) SEM images of ZN and ZP LDH nanoparticles; ZN showing flake-like morphology, whereas ZP has granular morphology.


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Figure 2: Modification of structural and surface properties of LDHs after intercalation of drug and then dispersing the LDH-drug conjugate into PCL matrix; (a) Powder X-ray diffraction patterns of pure PCL, pure ZN, ZN‒R and PN‒R, ‘⋆’ marks indicate the new set of basal reflections which originates from RH intercalated LDH phases; (b) TEM images of ZN‒R and PN‒R, inset shows the SAED patterns of ZN‒R and PN‒R; (c) Zeta potentials of the indicated samples; and (d) AFM topographs of ZN‒R, PCL and PN‒R with corresponding height profiles showing relative surface roughness.



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Figure 3: In vitro controlled release experiment and its underlying mechanism; (a) Cumulative drug release profile for raloxifene intercalated LDHs (ZN-R, ZC-R and ZP-R), raloxifene embedded in PCL matrix (PCL-RH) and PCL coated ZN-R (PN-R) in PBS at pH ~7.4 at 37 °C. The data points are plotted as mean ± SD values obtained from three different set of experiments; (b) Linear fitting of the drug release data to (i) modified Freundlich model and (ii) Korsmeyer–Peppas model; release profiles for raloxifene intercalated LDHs are better fitted by the modified Freundlich model, whereas release profiles for PCL-RH and PN-R are most suited from Korsmeyer–Peppas model; (c) Schematic representation of possible drug release mechanisms; (i) in drug intercalated LDHs, drug molecules can release through anion exchange process whereas (ii) in PN-R, drug molecules first release from LDH interlayer region to polymer matrix followed by a slow diffusion through the polymer matrix to the release media for detection.


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a.

Fe 2p

Fe 2p3/2

ZP-R ZP

Fe 2p1/2

Zn 2p3/2

726.02

712.57

Zn 2p

Zn 2p1/2

1023.97

ZP-R ZP

1047.17

1023.94

Intensity / a.u.

725.57

712.83

ZC-R ZC

725.33

711.72

725.76

711.71

ZN-R ZN

Intensity / a.u.

725.78

712.51

1047.12 1046.6

1023.2

1046.38 1023.68

ZN-R ZN

1046.80 1046.26

1023.16

725.16

ZC-R ZC

1023.3

711.18

720

730

1020

1030

b.

c. 331

Transmittance / a.u.

ZN-R 395

225 220

220

266 283

RH

364

ZN

278

PCL

ZN-R RH ZN

PN-R

3430

293

1050

d.

PN-R

223 228

1040

B.E / e.V.

B.E / e.V.

ZN-R

3435

RH

1728

1592 1172

1595 1255

3455

PCL ZN

1597

1260

1728

3468

Heat flow / a.u.

710

Heat flow / a.u.

700

Absorbance / a.u.

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


PCL PN-R

Tm= 59oC ∆H= 78 Jg-1

Tm= 64.4 oC ∆ H= 86 Jg-1

40

60

T / oC

80

100

Tm= 223.3 oC -1 ∆H= 68 Jg

Tm= 264.3 0C -1 ∆H= 85 Jg

1172 1630 1382

200

300

400

Wavelength / nm

500

4000

3000

2000

-1

1000

Wavenumber / cm

50

100

150

200

T / oC

250

300

Figure 4: Understanding the nature of interaction between drug and LDH nanoparticles; (a) Fe 2p and Zn 2p XPS spectra for pristine LDHs and drug intercalated LDHs. The vertical lines indicate the peak position/binding energy; (b) comparison of solid-state UV−vis spectra of pristine LDHs (ZN), pure drug (RH), drug intercalated LDHs (ZN‒R) and polymer nanohybrid (PN‒R); (c) FTIR spectra of the indicated samples showing change of peak position arising from interactions between the components; and (d) DSC thermograms of free drugs and drug intercalated LDHs, inset figure represents DSC thermograms for pure polymer and polymer nanohybrid.




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Figure 5: In vitro cytotoxicity, biocompatibility and cellular uptake analysis; (a) Comparative cell viability study of pure raloxifene, ZN-R, ZP-R, PCL-RH and PN-R against HeLa cells using MTT assay as a function of incubation time. Untreated cells are considered as control. The results are presented with mean ± standard deviation (SD) values obtained from three independent experiments; (b) Fluorescent microscopic images of AO/ EtBr staining of control, pure drug, drug intercalated LDHs, PCL-RH and PN-R showing relative number density of cells after treatment; (c) Relative cell adhesion values of the developed materials after 12 h incubation; (d) Phase contrast images of the HeLa cells grown on top of the indicated substrates after 12 h incubation; (e) Cellular uptake kinetics into HeLa cells under different incubation times. The observed red fluorescence (rhodamine-B) extracted from the cells in the presence of RH–RdB, PCL–RdB, ZN–RdB and PN– RdB treated with 100 µg ml−1 or equivalent amount of RdB. The results are presented in mean ± standard deviation (SD) values obtained from three independent experiments; (f) Fluorescence microscopic images showing the cellular uptake of rhodamine-B labeled samples into HeLa cells. Cells are exposed to 100 µg ml−1 or equivalent amount of RdB labeled particles showing various intensity of fluorescence depending on cellular uptake; and (g) Schematic illustration of cellular uptake mechanism considering same HeLa cell with indicated substrate with varying surface potential and roughness.



Scheme I: Schematics representation of cell adhesion for two different substrates which induce cellular uptake differentially leading to improved anticancer efficacy using polymer nanohybrid as drug delivery vehicle.


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Figure 6: In vivo controlled release of the drug and its underlying effects on organs; (a) Plasma RH concentration versus time profiles for RH, ZN-R, ZP-R and PN-R towards Charles Foster albino female rats (n =4) after intravenous administration at 10 mg drug / Kg body weight and equivalent amount in various delivery vehicles; Changes of biochemical parameters with time: (b) serum alanine aminotransferase (ALT) level; (c) aspartate aminotransferase (AST) level; (d) urea and ACS Paragon Plus Environment


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creatinine activity at the 7th day in Charles Foster albino female rats after intravenous administration at 10 mg drug / Kg body weight and equivalent amount of drug intercalated materials. Results are expressed as mean ± SD, n =4; (e) Histopathological examination using hematoxylin and eosin (H & E) staining to evaluate the toxicity of liver treated with control (PBS), pure RH, ZN-R, ZP-R and PN-R at different indicated time intervals. Significant lesions are found mainly on the liver from the pure drug (RH) treated group.


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Graphical Table of Content

Engineered Cellular Uptake and Controlled Drug Delivery Using Two Dimensional Nanoparticle and Polymer for Cancer Treatment

Sudipta Senapati, Rashmi Shukla, Yamini Bhusan Tripathi, Arun Kumar Mahanta, Dipak Rana and Pralay Maiti Drug has been intercalated within two-dimensional platelet to have sandwitched structure followed by its embedment in polymer for designing novel drug delivery vehicle with sustained release, greater bioavailability and advanced anti-tumor efficacy without any adverse effect as opposed to conventional chemotherapeutic treatment of cancer.


Engineered Cellular Uptake and Controlled Drug Delivery Using Two

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