Development of zoledronic acid-based nanoassemblies for bone

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Development of zoledronic acid-based nanoassemblies for bone-targeted anti-cancer therapy Elham Hatami, Prashanth K.B. Nagesh, Pallabita Chowdhury, Stacie Elliott, Deanna Shields, Subhash Chauhan, Meena Jaggi, and Murali M. Yallapu ACS Biomater. Sci. Eng., Just Accepted Manuscript • Publication Date (Web): 27 Mar 2019 Downloaded from http://pubs.acs.org on March 27, 2019

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ACS Biomaterials Science & Engineering

Development of zoledronic acid-based nanoassemblies for bone-targeted anti-cancer therapy Elham Hatami‡, Prashanth Kumar Bhusetty Nagesh‡, Pallabita Chowdhury, Stacie Elliot, Deanna Shields, Subhash Chand Chauhan, Meena Jaggi, and Murali Mohan Yallapu* Department of Pharmaceutical Sciences, College of Pharmacy, 881 Madison Ave, University of Tennessee Health Science Center, Memphis, TN-38163, USA

‡- Equally contributed to this work

To whom correspondence should be addressed Dr. Murali M. Yallapu Department of Pharmaceutical Sciences 881 Madison Avenue, Suite 447 Memphis, Tennessee 38163 Phone: (901) 448-1536 Fax: (901) 448-3446 E-mail: [email protected]

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Abstract Bone metastasis occurs in majority of cancer patients, which hampers quality of life and significantly decreases survival. Aggressive chemotherapy is a traditional treatment regimen which induces severe systemic toxicities. Therefore, bone-directed therapies are highly warranted. We report a novel nanoparticle formulation that is composed of poly(vinylpyrrolidone) and tannic acid core nanoparticles (PVT NPs) that forms self-assembly with zoledronic acid (ZA@PVT NPs). The construction of ZA@PVT NPs was confirmed by particle size, zeta potential, transmission electron microscopy, and spectral analyses. An optimized bone-targeted ZA@PVT NPs formulation showed greater binding and internalization in in vitro with metastasis prostate and breast cancer cells. ZA@PVT NPs were able to deliver ZA more efficiently to tumor cells, which inhibited proliferation of human prostate and breast cancer cells. In addition, ZA@PVT NPs were capable of targeting mouse bones and prostate tumor microarray tissues (ex vivo) while sparing all other vital organs. More importantly, ZA@PVT NPs induce chemo sensitization to docetaxel treatment in cancer cells. Overall, the study results confirm that ZA-based, bone-targeted NPs have great potential for the treatment of bone metastasis in the near future.

Key Words: nanoparticles, bone metastasis, breast cancer, prostate cancer, and lung cancer.


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Introduction Bone metastasis is commonly observed in patients with breast and prostate cancer (up to 70%), and carcinoma of lung, colon, stomach, kidney, and/or multiple myeloma (15-30%) 1-5. Bone is the third most prominent site (after lung and liver) for metastasis 6 due its environment, which engenders easy growth of tumors supported by nutrients, niche, and oxygen 7. Bone metastases often destroy the bone structure and cause fractures with clinical scenarios of severe pain, hypercalcemia, and catagma. Metastasized tumor cells reaching to the spine may lead to paralysis. Altogether, bone metastasis severely diminishes quality of life and is the cause of cancer-associated deaths. In the United States alone, there are about 569, 000 patients alive and experiencing severe fatal clinical illness (Oncology Services Comprehensive Electronic Records) 8.

Bone metastasis is a terminal state of cancer causing cancer-associated deaths 9. Current

therapy options to manage bone metastases is primarily palliative therapies and therefore there is tremendous clinical need to develop effective therapies to address such conditions.

In recent years, significant progress has been made in the management of bone metastasis. Advanced stage prostate cancer cells readily cause osteoblastic lesions but along excess of bone formation. In this condition, osteoblastic (OB) and osteolytic metastatic lesions are present. Bone marrow mesenchymal cells migrate towards regenerating tissues, primary tumors, and metastatic sites where they interact with tumor cells and helps for the tumor growth through the release of growth factors. Additionally, adipocytes from bone marrow can also promote cancer progression in prostate cancer. Bisphosphonates (BPs) were introduced in the 1970s as bone scanning agents 10-11

due to their ability to adsorb to bone mineral, i.e., hydroxyapatite (HA). Their parent and 3 ACS Paragon Plus Environment


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newer analogues continue to be employed in the treatment of osteoporosis, bone resorption, hypercalcemia of malignancy, myeloma, and bone metastases 12-13. Third generation BP analogues, alendronate sodium (Fosamax, Merck, 1999) and zoledronates (Reclast, Novartis, 2007) have been approved by FDA as anti-resorptive agents for their clinical use. Zoledronic acid aids in osteoblastic and adipocytic (AD) differentiation in MSCs and in osteoclast (OC) differentiation in monocytes which may disrupt micro-environmental interactions mediated by MSCs or adipocytes. A combination of bisphosphonate drug (to minimize bone pain) and chemotherapy agent (to control tumor growth) is commonly employed to tackle this disease 14-15. BPs themselves do not exhibit significant anti-cancer potential and the use of higher doses or continuous use may lead to osteonecrosis 16-17. Therefore, BPs are given along with conventional chemotherapy drugs. However, poor selectivity of conventional chemotherapy induces systemic toxicities.

A multifunctional nanomedicine that targets bone metastasis, supports the skeletal structure, and provides controlled release of an effective drug can prevent cancer growth and recurrence 18-19. Since zoledronic acid (ZA, Zometa®) exhibits up to 20–fold increased targeting of bone metastatic lesions than targeting of other major tissues, they can be serve as excellent functional ligands on nanoparticles. Bone-targeting drug delivery systems 20-21 such as polymer conjugates 22-24,

polymer nanoparticles 25-26, dendrimers 27, polymer micelles 28-30, liposomes 31-32, magnetic

nanoparticles 33-34, and silica nanoparticles 35-36 with bisphosphonate motif have been constructed. Among them, polymer and liposomal formulations have immense translational potential due to their inherent proven biocompatibility, longer circulation, and slow release of the encapsulated therapeutics 20-21. However, nanomedicines using liposomes are highly suitable for


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delivering potent molecules and may not be applicable for higher-targeting motif doses using the ligand in the current study, ZA. Considering this aspect, we approach a facile polymer selfassembly nanomedicine approach which may offer a solution for such interface diseases. In particular, we choose poly(vinylpyrrolidone) (PVP, also referred to as polyvidone), which is a synthetic water-soluble polymer, widely employed as a binder in many pharmaceutical tablets and eye drops. PVP is commonly found in over-the-counter medications.

Tannic acid (TA), a natural polyphenol molecule, has shown drug carrier properties 37-39. TA is known as coloring additive agent, pharmaceutical excipient, and a biocompatible and non-toxic compound according to the United States Food and Drug Administration. Its use as a therapeutic carrier has been tested for controlled drug release and improved therapeutic benefit in in vitro 4044

and in vivo mouse 39 models. Our long-term goal is to establish PVP and TA combination as a

base for nanoformulations for treatment of various chronic diseases including cancer. Conventional nanoparticle-based formulations follow a passive targeting mechanism i.e., the enhanced permeation and retention effect may not suitable due to the lack of leaky vasculature at the metastases bone sites 45-46. Zoledronic acid is a third-generation bisphosphonate drug molecule widely used clinically to minimize pain and skeletal related events in patients with bone metastases 47. This molecule comprises bisphosphonate which has strong binding potential with hydroxyapatite of the bone, thus it is a bone-directing molecule 48-49. Upon reaching the bone, ZA forms strong bonds with calcium, which are stable in bone for about a year. Therefore, purpose of the current study was to develop a structurally well-defined nanoparticle system for effective targeting of bone to treat bone metastasis. We synthesized a core nanoparticle formulation of poly(vinyl pyrrolidone) and tannic acid (PVT NPs) through self-assembly process



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where pendant pyrrolidone groups are assembled with hydroxyl groups of tannic acid. Based on these core nanoparticles, a series of bone-targeted nanoparticle formulations were generated by the mixing of PVT NPs with various concentrations of zoledronic acid (ZA@PVT NPs) by a cross-linking method. These nanoparticles were prepared using a self-assembly approach as reported earlier 50-51. Tannic acid is a universal molecule with drug binding capacity, and induces self-assembly and cross-linking formation with PVP and ZA, whereas ZA is primarily intended to impart better bone targeting capacity (Figure 1). Such bone-directed nanoformulations will be more beneficial than conventional chemotherapy to bone metastases patients.

Materials and Methods Cell culture Human prostate (C4-2 and PC-3) and breast (MDA-MB-231) cancer cell lines (which have bone metastasis potential or derived from bone metastasis) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). These cells have tendency to metastasize to bone. These cell lines were maintained in Roswell Park Memorial Institute medium (RPMI) or Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin (Thermo Fisher Scientific, Grand Island, NY). All cell lines were grown at 37°C in a humidified 5% CO2 atmosphere (Thermo Fisher Scientific, Waltham, MA, USA). Cellular uptake, proliferation, and colony formation experiments were performed at passages below 10.

Synthesis and physico-chemical characterization of ZA@PVT NPs


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Zoledronic acid coated poly(vinyl pyrrolidone)-tannic acid nanoassemblies were synthesized in sequential self-assembly and crosslinking approach. In brief, 100 μg PVP (100 μL) and 100 μg TA (100 μL) were dissolved in 800 μL milliQ water and stirred overnight to achieve a uniform self-assemblies i.e., PVT NPs. To this solution, various concentrations (0, 50, 100, 150, and 200 μg) of ZA was supplemented to coat on the assemblies to achieve a stable ZA@PVT NPs formulation. In order to induce strong crosslinks between PVT NPs and ZA a small portion of triol (glycerol, 0.1wt%) was employed. The final formulation was separated from unbound/assembled PVP, TA, and ZA by centrifugation at 10,000 rpm. The supernatant was removed and pellet containing ZA@PVT NPs were used for all physico-chemical characterization. Note: ZA (200 μg) containing ZA@PVT NPs were employed for all in vitro and ex vivo studies since ZA need to be higher in concentrations for inducing its anti-cancer potentials in bone metastasis cancer cells.

PVT NPs and ZA@PVT NPs were characterized by dynamic light scattering (DLS) (Nano ZS, Malvern Instruments, Malvern, UK), transmission electron microscopy (JEOL 200EX TEM, JEOL Ltd, Tokyo, Japan), Fourier transform infrared spectroscopy (Spectrum 100 FTIR spectrometer, Waltham, MA) to confirm composition and nanoparticle formation. All protocols and measuring parameters implemented for these measurements were performed as described in our previously published reports 40, 51-52. DLS measurements were performed for 50 µL of PVT or ZA@PVT nanoparticles which were dispersed in 1 mL deionized water and just after a quick probe sonication (VirSonic Ultrasonic Cell Disrupter 100, VirTis) for 30 s. Size measurements of particles were conducted at 25 °C for 2 min and data was reported from triplicate runs. The zeta potential of nanoparticles were acquired in 1× phosphate-buffered saline (PBS) solution (50 µL



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in 1 mL PBS). Data was acquired for 30 runs and presented an average of 3 readings. For TEM study, approximately 50 μL of nanoparticle solutions (prepared for DLS measurements) were placed on a shiny side of 150 mesh TEM grid (Electron Microscopy Sciences, PA, USA), subsequently stained using 2% (w/v) uranyl acetate solution, and imaged under the TEM system using an AMT camera at a direct magnification of 100,000×. The FTIR spectra were acquired on the Universal ATR sampling Accessory plate for lyophilized PVT and PVT@ZA nanoparticles between wavelengths of 4000 and 650 cm−1 at a scanning speed of 4 cm−1.

Cellular Uptake Study The cellular uptake of PVT NPs and ZA@PVT NPs that are encapsulated with a fluorescent dye (coumarin 6, C6) by prostate (C4-2 and PC3) and breast (MDA-MB-231) cancer cells were observed using microscopy and flow cytometry methods as described earlier 40, 51. For this, C6 dye (in acetone, 1 mg/mL) was added to the formulations along with 100 μg PVP (100 μL) and 100 μg TA (100 μL) with and without 0, 50, 100, 150, and 200 μg of ZA (as mentioned in Section 2.2) to achieve a stable C6 loaded PVT or C6 loaded ZA@PVT NPs formulations. At the end, unbound C6 dye was removed from supernatant by centrifugation of formulation at 5000 rpm for 10 min 53. It is possible that C6 can leached out from ZA@PVT NPs core. To minimize the leaching factors in our studies to investigate the early time points of 1, 3, and 6 h. We believe that even if the dye is leached out from nanoformulations, it can be observed as crystals because of precipitation which can be washed out by subsequent washes with 1X PBS. Coumarin 6 in PVT and ZA@PVT NPs can be viewed under a microscope and flow cytometer under green fluorescence channel. For the microscopy (cellular uptake visualization) method, cancer cells (0.5×106 per well) were seeded in 12-well cell culture plates. After cells adhered to the place


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surface, cells were incubated with 2.5, 5, and 10 μg/mL (C6 equivalent) of coumarin 6containing PVT NPs and ZA@PVT NPs (ZA is constant) solutions for 3 hours. The cells without treatment group was considered as control for this experiment. After the indicated time period, cells were processed in phenol red-free DMEM medium for imaging under EVOS® 214 FL Imaging System in a GFP channel (AMF4300, Life Technologies, Carlsbad, CA, USA). In flow cytometry method, under the same conditions, cells were prepared for semi-quantitative uptake estimation. After treatments, the cells were trypsinized and collected in 1 mL phenol red-free DMEM medium as reported previously. The uptake of coumarin 6-containing PVT NPs and ZA@PVT NPs was analyzed using NovoCyte Flow Cytometer (ACEA NovoCyte® 1000, ACEA Biosciences, Inc. San Diego, Ca, USA) in FITC channel (fluorescence measurements at λex: 485 nm and λem: 520 nm). The mean fluorescence intensity (MFI) and standard deviations were documented from triplicate measurements. Cellular Binding Study Cellular uptake of nanoparticles in live cells adopt number of cellular internalization pathways. Additionally, cellular uptake study cannot provide specific cellular binding capacity. Therefore, we studied cellular binding capacity of C6 loaded PVT NPs and ZA@PVT NPs on paraformaldehyde fixed prostate (PC3) and breast (MDA-MB-231) cancer cells. Since these are not live cells interaction of particles with cells are considered as cellular binding affinity. For this, briefly 0.5 x 106 cells were grown on coverslips in a 12-well cell culture plates. Next day, cells were fixed in 4% paraformaldehyde in PBS, pH 7.4 for 15 min at room temperature. After washing, cells were then incubated with 5 μg/mL C6 equivalent PVT NPs and ZA@PVT NPs for 6 hrs. Leaching of dye from the nanoformulations could be possible, for this three through washes with 1X PBS for 5 minutes each was performed after the incubation time. Followed by which DAPI 9 ACS Paragon Plus Environment


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staining was done to stain the nucleus for 45 minutes at room temperature. The coverslips were then mounted using Prolong Diamond Antifade Mountant (catalogue no. P36961, ThermoFisher Scientific) and imaged using a EVOS® 214 FL Imaging System in a GFP and DAPI channel (AMF4300, Life Technologies, Carlsbad, CA, USA). The quantification of the mean fluorescence intensity was quantified in comparison to the fold change with respect to the control cells for GFP channel. Fluorescent intensities were quantified using Image J. Data are represented by measuring fluorescent intensities from at least 100 cells in 3 separate images. A fluorescence based quenching assay was employed to further examine the ZA@PVT NPs (no C6 dye) cellular binding ability of cancer cells. In this study, the cell protein lysates from PC-3, and MDA-MB-231 cells were used to determine the binding affinity of PVT NPs and ZA@PVT NPs. The nanoparticles bind to the proteins and cause reduction in protein tryptophan residues. The extent of fluorescence quenching indicates superior interaction/binding of NPs with proteins or cells. For this, 100 µg protein lysate of each cell line was dispersed in 1 mL 1X PBS and used to interact with 10 µg PVT NPs or ZA@PVT NPs solution (no dye labelling here with NPs) in a 10 mm path length quartz cuvette (1.5 mL) by titration method. The fluorescence measurements accumulated using a SpectraMax Plus plate reader (Molecular Devices, Sunnyvale, CA, USA) in the wavelength range of 250–450 nm at an excitation wavelength of 280 nm. Proliferation assay The cell growth inhibition potential of ZA nanoparticles was determined using a cell proliferation kit (CellTiter 96® AQueous One Solution, MTS reagent, Promega Corporation, Madison, WI, USA) 51. For this study, cancer cells (5 × 103) were seeded in 100 μL of medium into a 96-well plate. After 24 hours, cells were treated with indicated concentrations of ZA or ZA@PVT NPs for 48 hours. PVT NPs used as control to see whether they induce any toxicity on 10 ACS Paragon Plus Environment

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these cells. A 20 μL MTS reagent was added in each well to existing medium and allowed to develop color due to formazan crystals formation for 3 hours. The intensity of the absorbed color of intracellular formazan was measured at 490 nm using a microplate reader (Cytation™ 5, BioTek Instruments, Winooski, VT, USA). The percentage of cell growth was calculated with the formula: [𝑇ℎ𝑒 𝑎𝑏𝑠𝑜𝑟𝑝𝑡𝑖𝑜𝑛 𝑜𝑓 𝑡ℎ𝑒 𝑡𝑟𝑒𝑎𝑡𝑒𝑑 𝑐𝑒𝑙𝑙𝑠/ 𝑡ℎ𝑒 𝑎𝑏𝑠𝑜𝑟𝑝𝑡𝑖𝑜𝑛 𝑜𝑓 𝑡ℎ𝑒 𝑢𝑛𝑡𝑟𝑒𝑎𝑡𝑒𝑑 𝑐𝑒𝑙𝑙𝑠] × 100 From the same study, cells were imaged under EVOS® 214 FL Imaging System in a Phase Contrast mode (AMF4300, Life Technologies, Carlsbad, CA, USA) for a comparative analysis with proliferation assay. Similarly, we demonstrate interaction of docetaxel (Dtxl) that is encapsulated in the ZA@PVT NPs to enhance cytotoxicity in comparison to the free Dtxl using proliferation assay. For this, cancer cells (5 × 103/well) were seeded in 96-well plates. Next, cells were treated with 0–10 nM free Dtxl and 0-10 nM equivalent Dtxl loaded ZA@PVT NPs (Dtxl loading was confirmed by HPLC as 945 µg/10 mg of ZA@PVT NPs)37.

Immunofluorescence The ex vivo specific tumor targeting behavior of ZA@PVT NPs was examined on tissue microarrays (TMA) slides (US Biomax, Inc., Derwood, MD USA) according to our earlier report 54.

The slides were of 24 paraffin-embedded tissue samples (n = 12 non-neoplastic, n = 6

moderately differentiated, and n = 6 poorly differentiated). The targeting ability of nanoparticles was tested on the TMA slides, which were tracked using dye in nanoparticles. In this process, TMA slides were first subjected to heat-induced antigen retrieval and an immunofluorescence procedure was followed employing Biocare Kit (Biocare Medical, Concord, CA). The tissue sections were placed at 60oC for 15 min. Then TMA slides were processed to remove paraffin



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(placed in xylene at room temperature for 15 min), rehydrated (sequentially in in an ethanol series of 100%, 90%, and 70%, 5 min each), treated with 0.3% hydrogen peroxide (to block endogenous peroxidase), and rinsed in deionized water and stored in PBST. Afterwards, TMA slides were blocked with normal donkey serum (5% in PBST, Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 hour in order to avoid non-specific binding. Under wet conditions, these TMA slides were probed with 25 μg (C6 equivalent) of C6-containing PVT (un-targeted NPs) or ZA@PVT NPs (targeted NPs) at 4°C. Note: C6 may leach out from ZA@PVT NPs, however, leached dye form precipitate on tissue slide which were removed by through washing with 1X PBS. Additionally, loosely bound PVT NPs or ZA@PVT NPs were removed during this step which would leave only strongly bound NPs. TMA slides were then washed with 1 × PBS (three times, 15 min each). Finally, TMA slides were counter stained with 4′,6-diamidino-2-phenylindole (DAPI) containing VECTASHIELD antifade mounting medium (Vector Labs, Burlingame, CA). The green fluorescence arose from C6 NPs indicating the targeting ability. This was imaged and quantified using a laser scanning confocal microscope (Carl Zeiss LSM 710 , Oberkochen, Germany). For quantification purpose, the corrected total cell fluorescence (CTCF) was calculated at four different fields using ImageJ Software (US NIH, http://imagej.nih.gov/ij/, 1997–2015) using the equation: CTCF = Integrated Density - (Area of selected cell × Mean fluorescence of background readings).

Ex vivo bone and tumor targeting All procedures were approved by the guidelines of the Institutional Animal Care and Use Committee of the University of Tennessee Health Science Center]. The UTHSC animal facility donated dead animal organs (with fixative) for this study. The parts of the leg used in the study


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were excised, then the muscles and residual tissues surrounding the femurs were removed. The cleaned leg femurs were used to determine the ex vivo bone targeting effects of ZA@PAPT NPs. These were incubated with indocyanine green (ICG)-loaded PVT NPs and ZA@PVT NPs to monitor their targeted efficacy. 50 μg equivalent of ICG-loaded PVT NPs or the same amount of targeted ICG-loaded ZA@PVT NPs was employed (2 mL). After 24 hrs incubation with these nanoparticles, the animal legs were washed and imaged using an in vivo IVIS Spectrum Imaging System (Caliper Life Sciences). A long-pass excitation filter at 690 nm attached with a coupled device camera (CCD) was employed for fluorescence imaging of tissues in 1×PBS arranged on Pretty dish. The spectral analyses were carried out at a fixed exposure time (5000 ms) while background was eliminated. The fluorescence signal intensity (measured as photon/s/cm2/sr) on images was quantified within the region of interest. In another set, 100 μg equivalent of ICGloaded PVT NPs or the same amount of targeted ICG-loaded ZA@PVT NPs was employed for bone targeting. For this, mice organ tissues (tumor, leg bone, heart, liver, lung, spleen and kidney) and PC-3 xenograft tumors were utilized to determine the targeting ability of ZA@PVT NPs. The ex vivo bone and tumor targeting potential of ICG-loaded PVT NPs and ZA@PVT NPs were determined by incubating the nanoparticles for 6 h.

Statistical analysis All reported biological assays were performed at least three times. Both in vitro and ex vivo experiments reported numerical quantifications as mean ± standard error of mean (SEM). The statistical significance of the data was calculated by a two-tailed Student’s t-test. A P value < 0.05 represents statistically significance. All statistical analyses were performed using GraphPad Prism 5.03 Software (GraphPad Software, San Diego, CA).



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Results ZA self-assembly leads to formation of nanoparticles For targeted delivery applications, the targeting motif needs to be immobilized efficiently on the surface of the nanoparticles via an appropriate method of conjugation. In this case, we choose TA as an interface between the PVP core and the ZA motif. TA can bind ZA molecules through physical interactions. However, it was noticed that this interaction suffers from low stability and burst release of ZA, hence we applied 0.1% glycerol as crosslinking agent. The introduction of tannic acid biointerface layers has received much attention recently in the drug delivery field 5558.

These layers in nanostructures impart unique properties including but not limited to long-term

biocompatibility, low toxicity, and a lack of immune stimulation. More importantly, TA has been employed as a pharmaceutical and food ingredient, and offers enhanced solubility profiles to various anticancer drugs37-39. In this study, we report solvent evaporation and simultaneous extrusion of self-assemblies of ZA@PVT NPs that were collected following the procedures developed for our previous protocols 40, 51. TA and PVP polymer exhibited particles size of formulation of ~ 430±15 nm (Figure 2A) with zeta potential of ~ - 5.8±0.4 mV (Figure 2B). Addition of ZA into these selfassemblies induce increase their particle sizes (532±65 to 522±57) due to deposition of ZA layers on PVT NPs. At the same time, zeta potential of ZA@PVT NPs decreased to - 5.89±0.6 to -10.6±0.6 with addition of various ZA concentrations (Figure 2B). Importantly, we also demonstrated that this composition does not change in particle size over a period of one week, indicating suspension and physical stability of the formulation (Supporting Information 1, Figure S1). Additionally, we show ZA@PVT NPs demonstrate spherical structure while maintaining its


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parent core of PVT NPs spherical morphology (Figure 2C). Since ZA allows the formulation to be directed to bone complex and effectively binds bone complex, we believed that ZA on nanoparticles can contribute to the targeting of bone tissue or bone metastasis in a similar way. Overall, this result discloses the feasibility of the self-assembly of TA, PVP, and ZA to form physically bound matrices collectively to generate nanoparticle formulation (Figure 1). The transmission electron microscope images of the ZA@PVT NPs are monodisperse and have round shape morphology in its dried state (Figure 1C). Their dried particle sizes were in the range of 54 to 116 nm (Figure 1C).

The FTIR spectra of pure PVP (aqua color) (-OH stretching vibrations 3430 cm-1, C-H asymmetric stretching vibrations 2962 cm-1, C=O stretching vibration 1663 cm-1, CH2 bending 1428 cm-1, C-N vibrations 1285 and 1016 cm-1) and TA (black color) (aromatic hydroxyl vibrations 3410 cm-1, C-H vibrations 2925 cm-1, aromatic C-O vibrations 1620 cm-1, aromatic CH stretchings 1420 cm-1, C-O vibrations 1050 and 805 cm-1) components that were used in nanoparticle generation is presented in Figure 2D. The FTIR spectra of PVT- core nanoparticles (blue color) (Figure 2D) exhibited a broad band in the range of 3400–3200 cm−1 which belongs to O-H stretching vibration of pyrrolidone groups of PVP and hydroxyl groups of TA. The increased peak in this range proves the presence of strong hydrogen bonding PVP and TA. In addition, PVT NPs exhibited strong presence of C=O/C-O vibrations at 1663/1620 cm-1, CH2 bending at 1428 cm-1, and C-O vibrations at 1050 cm-1 due to PVP and TA presence. Whereas in the case of ZA@PVT NPs, presence of all the above characteristic peaks are presented (Figure 2D, green color) and appears that ZA (Figure 2D, red color) and PVT NPs (Figure 2D, blue



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color) appears are overlapped. These data overall suggest that ZA@PVT NPs are constructed with PVP, TA, ZA.

ZA@PVT NPs promotes binding to bone and tumor cells Bone metastasis therapy regimens are associated with severe side effects due to non-specific drug actions on normal cells and tissues. Therefore, we intended to evaluate the functional targeting status of our nanoparticles using an ex vivo bone targeting assays. As expected and in accordance with existing literature that teaches when ZA is decorated on nanoparticles, this formulation has a strong affinity to bone (Figure 3A) due to its hydroxyl and phosphonate groups. The ZA@PVT NPs exhibited a superior binding affinity with HA compared to non-ZA nanoparticles. This increased affinity was noticed to have a nearly 2.1-fold increase over non-ZA material (Figure 3B). This data confirm that ZA NPs have proven their specificity and bonetargeting efficiency. Additionally, ex vivo study determines the superior accumulation of ZA@PVT NPs not only to the bone but also on PC-3 tumors compared to other mouse organs such as, heart, liver, lung, kidney, and spleen (Figure 3C-D). Whereas PVT NPs do not show significant bone targeting but it accumulates on PC-3 tumors due to the presence of tannic acid. PVT NPs and ZA@PVT NPs containing tannic acid layers are known inhibitors of many oncogenic proteins such as EGFR, CXCR4, FASN, etc., 59-61. As shown in Figure 3C, the fluorescence signals of ICG-loaded PVT NPs and ICG-loaded ZA@PVT NPs was negligible in other organs compared to bone and tumor. This provides insights the ability of ZA@PVT NPs to target both bone and tumor cells which can be useful for therapeutic perspective.

ZA motif on nanoparticles exhibits binding and internalization capacity in cancer cells


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We next examined whether ZA has a role in cellular uptake and internalization with cancer cells in vitro. In this study, cellular binding and uptake of ZA in cancer cells were studied with (ZA@PVT NPs) and without ZA-containing (PVT NPs) nanoparticles in PC-3 and MDA-MB231 cancer cells. To visualize targeted binding of nanoparticles, we encapsulated a C6 dye according to our previous protocols. Cellular images showed that ZA@PVT NPs collectively target cancer cells more effectively that non-ZA-containing nanoparticles in a dose-dependent manner (Figure 4A&D). Flow cytometry semi-quantitative assay was further confirmed such superior targeting ability of ZA@PVT NPs compared to PVT NPs (Figure 4B&E) in a pool of 10,000 cells. The quantification was presented in Figure 4C&F to confirm its significant uptake with ZA@PVT NPs. Together, this study indicates that ZA@PVT NPs bind to cancer cells at a 2.3-fold higher rate compared to PVT NPs. This assay visualized that ZA nanoparticles significantly bind and bundle tumor tissues than is found with normal or adjacent tumor tissues (Fig. 4D–F). Since cellular internalization of NPs is not measure of cellular specific binding to cancer cells, we further evaluated binding profiles of C6 loaded PVT NPs and C6 loaded ZA@PVT NPs on a paraffin fixed cancer cells. This data demonstrates a significant amount of ZA@PVT NPs binds to the cells compared to PVT NPs in confocal microscope (Figure 5A). Its quantification was presented in Figure 5B. The extent of ZA@PVT NPs binding with cancer cell lysates was examined by instantaneous fluorescence protein quenching method 53. This instantaneous cellular protein binding ability of ZA@PVT NPs is significant over PVT NPs (Figure 5C). This is due to combination of ZA and tannic acid present in ZA@PVT NPs which may help for superior cellular binding (Figure 5D). Together, these cellular binding and targeting results demonstrate the concept that ZA nanoparticles are capable of targeting cancer cells both in vitro and ex vivo.



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ZA@PVT NPs target prostate tumor tissues Next, we examined whether our ZA@PVT NPs have specific targeting capacity to target those metastatic potential cells. We used tissue microarrays (TMAs) of prostate cancer (3 cores of normal adjacent prostate cancer tissue, Stage II, III, and IV cancer tissues). To visualize targeted binding of nanoparticles, we encapsulated a C6 dye. Representative images of the binding of ZA@PVT NPs on normal tissues verses tumor tissues were presented in Figure 6A. A distinct targeted profile of ZA@PVT NPs binding was observed on these prostate tumor tissues compared to normal tissues. Dye binding on tissues was quantified using Image J software, and representative images of cores are presented in Figure 6A. There is strong evidence from fluorescence observation in tumor tissues vs normal tissues (Figure 6B). Both images and quantitative data of C6 intensities representing staining increase was observed, which is significantly very high at stage III and stage IV cancer tissues which are considered as metastasized tumors.

ZA@PVT NPs increase anti-cancer efficiency in cancer cells Since zoledronic acid has a higher bone-binding affinity and anti-cancer activity against cancer cells, surface modification of nanoparticles with this ligand can enhance binding to the bone surface, which can be used to treat bone metastasis diseases in a targeted manner. Therefore, in this work we employed ZA as a bone-targeting ligand. So far, we confirmed that ZA nanoparticles have targeted affinity to cancer cells, but whether these nanoparticles preserve or increase their efficacy is unknown. To validate this, proliferation assay was employed. In the proliferation assay, both free ZA and ZA@PVT NPs have shown dose-dependent therapeutic


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activity on three cancer cell lines (Figure 7A) which have metastasis potential to bone. However, superior anti-proliferative activity was noticed with ZA@PVT NPs. Further, this phenomenon is evident from their cellular images. Compared to free drug (ZA), ZA@PVT NPs demonstrated effective effects of inducing apoptotic features in cancer cells (Figure 7B) while ZA alone able to inhibit proliferation. Collectively, these results show that ZA@PVT nanoparticles are capable of delivering therapeutics and inducing enhanced therapy activity in cancer cells in vitro. Additionally, it is possible to encapsulate docetaxel in these ZA@PVT NPs which has encapsulation capacity of 945 µg/10 mg of ZA@PVT NPs. Moreover, docetaxel encapsulated ZA@PVT NPs significantly reduce docetaxel concentration to act on prostate cancer cells (Figure 8A). At all tested concentration ZA@PVT NPs exhibited superior anti-cancer activity on cancer cells compared to free docetaxel. This behavior can be confirmed from their cellular images (Supporting Information 2, Figure S2). Docetaxel show 50% cell inhibitory concentration (IC50) 9.09±0.1, 5.40±0.1, 9.04±0.1 nM in C4-2, PC-3, and MDA-MB-231 cells which was reduced to 3.11± 0.1, 2.20± 0.2, 2.40± 0.1 nM when used Dtxl loaded ZA@PVT NPs (Figure 8B).

Discussion A majority of breast and prostate cancer patients, and more than 20% of multiple myeloma, lung cancer, and other cancer sufferers experience bone metastasis. Upon metastasizing to bone, cancer cells mutually track within the bone matrices and interact with osteocytes for their growth and by protecting the tumor thus develops drug resistance. Such bone metastasis is an emergence that is highly difficult to treat and presents an unmet clinical need. Therefore, developing therapeutic strategies that rely on bone targeting components would minimize systemic side



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effects. Many studies confirm that third generation bisphosphonate drugs help to enrich bonedirected delivery of therapeutics to minimize bone metastasis-associated pain. However, these efforts help to minimize pain but also impose additional complex side effects. Our objective is to generate a therapeutic regimen with a bone-targeting nanoparticle carrier, which can be offer enhanced delivery of chemotherapy. Among many bisphosphonates (clodronate, etidronate, risedronate, ibandronate, alendronate, pamidronate, tiludronate, and zoledronate), zoledronate exhibits a stronger affinity for natural bone (80.8 µM) and HA (0.29 µM), similar to alendronate 62-63. Whereas all other mentioned bisphosphonates show 806, 90.7, 84.6, 116, 60.9, 173, 82.7 µM affinity for natural bone while 1.39, 0.84, 0.46, 0.42, 0.34 µM, not determined, not determined, for hydroxyapatite, respectively. Based on this bone-targeting affinity of ZA, nanoparticles containing ZA can be used as molecular probes, for drug delivery, or as an imaging agent for cancer microcalcification, atherosclerosis, and calcium urolithiasis.

Over the past two decades, there has been enormous progress in delivery therapeutics through nanomedicine, i.e., nanoparticle-mediated delivery of drugs. Successful nanoparticle formulation overcomes some conventional limitations of drugs by enhancing drug availability, lower drug toxicity, while improving cellular interactions, stability, and sustained drug release characteristics, while sparing normal cells. Until now, most nanomedicine uses a positive targeting route to target tumors via enhanced permeation and retention properties. However, a major drawback of developing nanomedicine for bone metastasis is the loss of vasculature in bone microenvironment. Thus, developing therapy that has specific action at the bone interface while avoiding interaction with normal cells is highly sought after. A bone metastasis targeting


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formulation that composed of PLGA with docetaxel and ZA would exhibits prolonged blood circulation half-life and reduced liver uptake, and cause significant higher retention at the bone site while tumor specific accumulation 64. Another nanoparticle-based formulation consists of PLGA, PEG with bisphosphonate reported to have specific targeting bone in mice studies. These two well-known formulations aim to target bone and achieve enhanced tumor retention. In contrast, we have developed a self-assembled nanoparticle formulation which is composed PVP, TA and ZA. The novelty and primary advantage of our formulation is due to tannic acid layers which provides significant tumor cells binding and inhibits various oncogenic signaling pathways59-61. Additionally, it is widely accepted that soft nanoparticles generated by polymeric chains builds in a longer circulation half-life, which may have tumor targeting capacity. Earlier studies demonstrated that movement of 125I PVP (average molecular weight 35,000) was capable of passing extravascular tissue fluid in bone 65. Systemic administration of nanoparticles would be able to target the bone tissue by crossing the blood-bone marrow barrier due to the vasculature in bone 66. Therefore, we expect that ZA@PVT NPs generated in this study will have a similar in vivo outcome. We confirm the suitability of this formulation for cancer therapeutics based on its particle size and zeta potential (Figure 2 and Supporting Information 1, Figure S1). This particle size ranges have been known to be capable of providing depot to tumors because of their longer circulation half-life, and avoidance of opsonization (renal or lymphatic clearance). Additionally, low negative zeta potential is an indicator for a safer formulation, whereas positive zeta potential is considered toxic.

ZA is a third-generation phosphonate source and its presence in nanoformulations would enhance their affinity to bones or bone metastasis. To further verify the specificity of ZA



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nanoparticles targeting cancer cells, we performed an immunofluorescence binding study on a tumor micro array which contains both tumor and normal or adjacent tumor tissues. Our study provides evidence for a successful nanoparticle assembly with ZA that promotes targeting bone and tumors than other vital organs (Figure 3&6). In addition, these assemblies efficiently and specifically targeted human cancer cells and cancer tissue cells compared to normal tissue cells (Figure 4-6). Various studies have demonstrated the antitumor effects of zoledronic acid 15, 67-68. The primary mechanism is inhibition of bone resorption, while there is ample evidence for its direct role in inhibiting cancer cell proliferation and inducing apoptosis 69-70. This also includes apoptosis of osteoclasts as well as cancer cells by activation of caspases to induce apoptosis. Furthermore, the ZA on these nanoparticles was able to induce anti-cancer activity in cancer cells more effectively than did free ZA (Figure 7).

Docetaxel is a commonly employed chemotherapy for bone metastasis that originates from breast or prostate cancer, which has a meaningful therapeutic benefit. However, many clinical studies suggest that severe systemic toxicity results from higher dosing regimens. Additionally, cancer cells adopt resistance phenomenon to these drugs due to overexpression of various multidrug resistance proteins. Delivery of these agents using bone-targeted nanoparticles may efficiently target metastasized tumor cells and induce significant antitumor properties. Although our work has not specifically examined the role of docetaxel nanoformulations with ZA ligand in mouse models, but ZA@PVT NPs boost the docetaxel efficacy in cell line models (Figure 8). However, given our nanoparticle tumor homing capabilities preserves activity of docetaxel over a period of time. Our future studies will delineate the specific role of nanoassemblies and how they are implicated in achieving enhanced anti-cancer activities.


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Overall, our data is a clinically relevant outcome since docetaxel and ZA have been used clinically for decades. The clinical evaluation of our nanoformulations would be a step closer to being developed as a therapeutic regimen with superior effects over free drug regimens in relevant animal and human clinical trials. Indeed, it is important to note that both drugs currently in clinical use induce severe toxicities. Therefore, developing regimens with either improved therapeutic benefits or those minimizing systemic side effects will be a major breakthrough in the clinical setting. This work reinforces the additional use of this formulation for a localized delivery of components that are required to maintain bone structure and bone health in bone regeneration research. To confirm the superior efficacy and targeting of this nanoformulation, it needs to be further verified in clinically relevant mouse models. Upon such verification, these nanoparticles can be proposed as a promising bone-targeted formulation to treat bone metastatic cancer.

Conclusions Collectively, our study demonstrated a facile approach to construct PVT NPs using physical self-assemblies of poly(vinylpyrrolidone), tannic acid, and zoledronic acid. The developed ZA@PVT NPs shows spherical structures and can be loaded with chemotherapy drugs in the self-assembled network. ZA@PVT NPs are efficient in targeting cancer cells in vitro, ex vivo, and have exhibited superior dose-dependent anti-cancer activity against prostate and breast cancer cells. The bone-targeting efficiency of PVT NPs was confirmed by 2.1-fold on mouse bone over that of PVT NPs (non-ZA-containing nanoparticles). Additionally, ZA@PVT NPs offers



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superior docetaxel delivery to tumor cells. We anticipate that these nanoparticles can open new bone-targeted drug delivery strategies to regulate bone metastases.

Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Stability of ZA@PVT NPs formulation measured by DLS and phase contrast images of cancer cells upon treatment with ZA@PVT NPs.

Acknowledgments The authors acknowledge the Core facilities at the College of Pharmacy and Center for Cancer Research, The University of Tennessee Health Science Center, Memphis, TN. The Department of Chemistry and Nebraska Center for Materials and Nanoscience analytical facilities are acknowledged for providing the thermal and XRD scans for our samples. This work was partially supported by the National Institutes of Health (K22 CA174841 and R15 CA 213232), University of Tennessee Health Science Center Funding: CORNET grant by Office of Research, and start-up & seed grant(s) by the College of Pharmacy, to Dr. Murali M. Yallapu. This work was also partially supported by grants obtained by Dr. Subhash C. Chauhan from the National Institutes of Health (R01 CA210192, R01 CA206069, and R01 CA204552).


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54. Nagesh, P. K. B.; Johnson, N. R.; Boya, V. K. N.; Chowdhury, P.; Othman, S. F.; KhalilzadSharghi, V.; Hafeez, B. B.; Ganju, A.; Khan, S.; Behrman, S. W.; Zafar, N.; Chauhan, S. C.; Jaggi, M.; Yallapu, M. M., PSMA targeted docetaxel-loaded superparamagnetic iron oxide nanoparticles for prostate cancer. Colloids and surfaces. B, Biointerfaces 2016, 144, 8-20. DOI: 10.1016/j.colsurfb.2016.03.071. 55. Lau, H. H.; Murney, R.; Yakovlev, N. L.; Novoselova, M. V.; Lim, S. H.; Roy, N.; Singh, H.; Sukhorukov, G. B.; Haigh, B.; Kiryukhin, M. V., Protein-tannic acid multilayer films: A multifunctional material for microencapsulation of food-derived bioactives. Journal of colloid and interface science 2017, 505, 332-340. DOI: 10.1016/j.jcis.2017.06.001. 56. Kozlovskaya, V.; Xue, B.; Lei, W.; Padgett, L. E.; Tse, H. M.; Kharlampieva, E., Hydrogenbonded multilayers of tannic acid as mediators of T-cell immunity. Advanced healthcare materials 2015, 4 (5), 686-94. DOI: 10.1002/adhm.201400657. 57. Adatoz, E. B.; Hendessi, S.; Ow-Yang, C. W.; Demirel, A. L., Restructuring of poly(2-ethyl2-oxazoline)/tannic acid multilayers into fibers. Soft matter 2018, 14 (19), 3849-3857. DOI: 10.1039/c8sm00381e. 58. Peng, L.; Cheng, F.; Zheng, Y.; Shi, Z.; He, W., Multilayer Assembly of Tannic Acid and an Amphiphilic Copolymer Poloxamer 188 on Planar Substrates toward Multifunctional Surfaces with Discrete Microdome-Shaped Features. Langmuir : the ACS journal of surfaces and colloids 2018, 34 (36), 10748-10756. DOI: 10.1021/acs.langmuir.8b01982. 59. Chen, X.; Beutler, J. A.; McCloud, T. G.; Loehfelm, A.; Yang, L.; Dong, H. F.; Chertov, O. Y.; Salcedo, R.; Oppenheim, J. J.; Howard, O. M., Tannic acid is an inhibitor of CXCL12 (SDF-1alpha)/CXCR4 with antiangiogenic activity. Clinical cancer research : an official journal of the American Association for Cancer Research 2003, 9 (8), 3115-23. 60. Fan, H.; Wu, D.; Tian, W.; Ma, X., Inhibitory effects of tannic acid on fatty acid synthase and 3T3-L1 preadipocyte. Biochimica et biophysica acta 2013, 1831 (7), 1260-6. 61. Darvin, P.; Joung, Y. H.; Kang, D. Y.; Sp, N.; Byun, H. J.; Hwang, T. S.; Sasidharakurup, H.; Lee, C. H.; Cho, K. H.; Park, K. D.; Lee, H. K.; Yang, Y. M., Tannic acid inhibits EGFR/STAT1/3 and enhances p38/STAT1 signalling axis in breast cancer cells. Journal of cellular and molecular medicine 2017, 21 (4), 720-734. DOI: 10.1111/jcmm.13015. 62. Leu, C. T.; Luegmayr, E.; Freedman, L. P.; Rodan, G. A.; Reszka, A. A., Relative binding affinities of bisphosphonates for human bone and relationship to antiresorptive efficacy. Bone 2006, 38 (5), 628-36. DOI: 10.1016/j.bone.2005.07.023. 63. Nancollas, G. H.; Tang, R.; Phipps, R. J.; Henneman, Z.; Gulde, S.; Wu, W.; Mangood, A.; Russell, R. G.; Ebetino, F. H., Novel insights into actions of bisphosphonates on bone: differences in interactions with hydroxyapatite. Bone 2006, 38 (5), 617-27. DOI: 10.1016/j.bone.2005.05.003. 64. Ramanlal Chaudhari, K.; Kumar, A.; Megraj Khandelwal, V. K.; Ukawala, M.; Manjappa, A. S.; Mishra, A. K.; Monkkonen, J.; Ramachandra Murthy, R. S., Bone metastasis targeting: a novel approach to reach bone using Zoledronate anchored PLGA nanoparticle as carrier system loaded with Docetaxel. Journal of controlled release : official journal of the Controlled Release Society 2012, 158 (3), 470-8. DOI: 10.1016/j.jconrel.2011.11.020. 65. Owen, M.; Howlett, C. R.; Triffitt, J. T., Movement of 125I albumin and 125I polyvinylpyrrolidone through bone tissue fluid. Calcified tissue research 1977, 23 (2), 10312. 66. Howlett, C. R.; Dickson, M.; Sheridan, A. K., The fine structure of the proximal growth plate of the avian tibia: vascular supply. Journal of anatomy 1984, 139 ( Pt 1), 115-32. 29 ACS Paragon Plus Environment


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67. Berenson, J. R., The Medical Research Council Myeloma IX Trial: new clinical insights on the anticancer effects of zoledronic acid in patients with multiple myeloma. Clinical lymphoma, myeloma & leukemia 2012, 12 (1), 2-4. DOI: 10.1016/j.clml.2011.03.025. 68. Clyne, M., Prostate cancer: New light shed on the anticancer effects of zoledronic acid. Nature reviews. Urology 2012, 9 (5), 235. DOI: 10.1038/nrurol.2012.70. 69. Koul, H. K.; Koul, S.; Meacham, R. B., New role for an established drug? Bisphosphonates as potential anticancer agents. Prostate cancer and prostatic diseases 2012, 15 (2), 111-9. DOI: 10.1038/pcan.2011.41. 70. Gnant, M.; Clezardin, P., Direct and indirect anticancer activity of bisphosphonates: a brief review of published literature. Cancer treatment reviews 2012, 38 (5), 407-15. DOI: 10.1016/j.ctrv.2011.09.003.


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Captions of the Figures Figure 1. Construction ZA modified poly(vinyl pyrrolidone)/tannic acid (ZA@PVT) nanoparticles for bone targeting. A) Chemical Structures of PVP, TA, and ZA that were employed to generate ZA@PVT NPs. B) Hypothetical structure and sequential self-assembly and cross-linking approach followed to produce ZA@PVT NPs formulation. Figure 2. Physico-chemical characterization of ZA@PVT NPs. A-B) particle size and zeta potential of PVT and ZA@PVT nanoparticles. Particle size measurements were presented from triplicate runs (2 min each run) and zeta potential of nanoparticles were represented from an average of 3 readings of 30 runs on Malvern Nano ZS. Mean ± SEM (n = 3). C) Representative TEM image of PVT core nanoparticles and ZA@PVT nanoparticles (single emulsion), negatively stained with 2% (w/v) uranyl acetate solution, imaged at 80.0 kV with the help of AMT camera at a direct magnification of 100,000×. Scale bar: 500 nm. D) FTIR spectra of TA, PVP, ZA, PVT, and ZA@PVT NPs. The spectra is presented from 4000 to 650 cm−1 with a resolution of 4 cm−1 and an average of 32 scans for each sample. Figure 3. Bone-targeting ability of ZA@PVT NPs. Ex vivo bone-homing ability of ZA@PVT NPs. IVIS Spectrum epi-fluorescence image of bone fragment targeting after 24 hrs incubation with ICG-labeled PVT (nontargeted, left) or ICG-labeled ZA@PVT NPs (targeted, right). The image analyses were achieved at a fixed exposure time (5000 ms) while background was eliminated. The fluorescence signal intensity was reported on images as photon/s/cm2/sr within the region of interest. Scale range: Minimum = 4.77 e7 (red) and Maximum= 1.4 e9 (blue). B) Epifluorescence quantification were represented after treatment with ZA@PVT NPs that significantly targets bone in comparison to PVT NPs. Data reported as Mean ± SEM (n = 3) and *p < 0.05. C) A comparative ex vivo bone and tumor targeting ability of ZA@PVT NPs. IVIS Spectrum epifluorescence image of tissue targeting ability after 5 min incubation with ICG-labeled PVT (nontargeted, left) or ICG-labeled ZA@PVT NPs (targeted, right). The image analyses were similar to other ex vivo study. D) Quantification of tissue targeting ability shown in Figure 3C. Data reported as Mean ± SEM (n = 3) and *p < 0.05. Figure 4. ZA improves ZA@PVT nanoparticles cellular internalization and uptake in vitro in PC-3 and MDA-MB-231 cells. Cancer cells (0.5×106 per well) were incubated with 5 μg/mL (C6 equivalent) of coumarin 6-containing PVT or ZA@PVT NPs solutions for 3 hours (ZA dosedependent). A-C) PC-3 and D-F) MDA-MB-231 cell lines. A&D) Representative fluorescence images of cellular uptake of fluorescently labeled PVT and ZA@PVT NPs were imaged under EVOS® 214 FL Imaging System in a GFP channel. Scale bar: 50 µm. Cells treated without dyeNPs or with no treatment were considered as control for this experiment. B-C&E-F) Quantification of uptake of fluorescently labeled PVT and ZA@PVT NPs were measured from NovoCyte Flow Cytometer in FITC channel (fluorescence measurements at λex: 485 nm and λem: 520 nm) analyzing 10,000 cells. Mean Fluorescence Intensity (MFI) reported as Mean ± SEM (n = 3) and *p < 0.05. Figure 5. ZA promotes cellular binding of ZA@PVT nanoparticles in PC-3 and MDA-MB231 cells. A-B) Cancer cells (0.5×106 per well) grown on coverslips in a 12-well cell culture plates 31 ACS Paragon Plus Environment


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and fixed with 4% paraformaldehyde. Then cells were incubated with 5 μg/mL (C6 equivalent) of coumarin 6-containing PVT or ZA@PVT NPs solutions for 6 hours. A) Representative fluorescence images of cellular binding of fluorescently labeled PVT and ZA@PVT NPs were imaged under EVOS® 214 FL Imaging System in a GFP channel. Scale bar: 50 µm. Cells treated without dye-NPs or with no treatment were considered as control for this experiment. B) Quantification of cellular binding of fluorescently labeled PVT and ZA@PVT NPs were measured as presented in section 2.4. Fluorescent intensities were quantified using Image J. Data are represented (Mean ± SEM, n = 3 and *p < 0.05) by measuring fluorescent intensities from at least 100 cells in 3 separate images. C-D) ZA@PVT NPs show superior binding affinity to cancer cell proteins. Fluorescence intensity decrease is an indication for interaction between protein lysates and PVT NPs and ZA@PVT NPs. D) Area under the curve (AUC) of fluorescence data of ZA@PVT NPs demonstrates statistical significance protein quenching but not with PVT NPs. Data represents Mean ± SE (n = 3). Figure 6. ZA@PVT NPs offer significant binding with metastasized tumors (Ex vivo). The ex vivo tumor targeting ability of ZA@PVT NPs were assessed using tissue microarrays (TMA) slides. After processing TMA slides as mentioned in protocol, slides were probed with 25 μg (C6 equivalent) of C6-containing PVT or ZA@PVT NPs and incubated at 4°C for an overnight, then washed with 1 × PBS and protected from fluorescence bleaching using VECTASHIELD antifade mounting medium. A) Representative and semi-quantification of C6-containing PVT or ZA@PVT NPs targeting were measured from the cellular binding using confocal microscope. The green fluorescence arose from C6 NPs indicating the targeting ability were imaged using Carl Zeiss LSM 710 laser scanning confocal microscope at a direct magnification of 40,000×. Scale bar: 50 µm. B) Quantitative fluorescence measurement of C6-containing PVT or ZA@PVT NPs binding to the tissues were quantified through CTCF intensity using ImageJ Software. Data represented from atleast 4 fields. Data reported as Mean ± SEM (n = 4) and *p < 0.05. Figure 7. ZA@PVT NPs exhibit superior in vitro anti-cancer effects on cancer cells. C4-2, PC-3, and MDA-MB-231 (5 × 103) cells were plated in 96-well and cells were treated after 24 hours with varying concentrations of ZA@PVT NPs for 48 hours. A-C) Proliferation inhibition: After treatment, 20 μL MTS reagent were added in each well to the medium already present and allowed to incubate for 3 hours which was later measured using a Cytation™ 5 microplate reader at 490 nm. The percentage of cell growth was calculated as mentioned in protocol. Data reported as Mean ± SEM (n = 6) and *p < 0.05. D) Visual evidence: Representative images of cells after each treatment group acquired under EVOS® 214 FL Imaging System in a Phase Contrast mode. Direct magnification of 20,000× and Scale bar: 200 µm. Figure 8. ZA@PVT NPs demonstrates significant anti-cancer effects on cancer cells. A) A representative TEM image of docetaxel loaded ZA@PVT NPs. direct magnification of 100,000×. Scale bar: 500 nm. Cancer cells (5 × 103) were plated in 96-well and cells were treated after 24 hours with varying docetaxel or equivalent docetaxel loaded concentrations of ZA@PVT NPs for 48 hours. B) Proliferation inhibition of docetaxel and docetaxel loaded ZA@PVT NPs in C4-2, PC-3, and MDA-MB-231 cells measured as mentioned in Figure 7 protocol. Data reported as Mean ± SEM (n = 6) and *p < 0.05. C) Calculated 50% cell inhibitory growth of cancer cells. (Supporting Information 2, Figure S2). Representative images of cells


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after each treatment group acquired under EVOS® 214 FL Imaging System in a Phase Contrast mode. Direct magnification of 20,000× and Scale bar: 200 µm.



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Table of contents (TOC) Graphic Development of zoledronic acid-based nanoassemblies for bone-targeted anti-cancer therapy Elham Hatami‡, Prashanth Kumar Bhusetty Nagesh‡, Pallabita Chowdhury, Stacie Elliot, Deanna Shields, Subhash Chand Chauhan, Meena Jaggi, and Murali Mohan Yallapu* Department of Pharmaceutical Sciences, College of Pharmacy, 881 Madison Ave, University of Tennessee Health Science Center, Memphis, TN-38163, USA


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Figure 1



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Figure 2


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Figure 3



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Figure 4


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Figure 5



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Figure 6


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Figure 7



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Figure 8


Development of zoledronic acid-based nanoassemblies for bone

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