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ATP-Decorated Mesoporous Silica for Biomineralization of Calcium Carbonate and P2 Purinergic ReceptorMediated Antitumor Activity against Aggressive Lymphoma Prateek Srivastava, Sumit Kumar Hira, Divesh N Narayan Srivastava, Vivek Kumar Singh, Uttam Gupta, Ranjeet Singh, Ram Adhar Singh, and Partha Pratim Manna ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b18729 • Publication Date (Web): 02 Feb 2018 Downloaded from http://pubs.acs.org on February 4, 2018

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ACS Applied Materials & Interfaces

ATP-Decorated

Mesoporous

Silica

for

Biomineralization of Calcium Carbonate and P2 Purinergic Receptor-Mediated Antitumor Activity against Aggressive Lymphoma Prateek Srivastava,†,‡,§ Sumit Kumar Hira,†,⊥ Divesh Narayan Srivastava,|| Vivek Kumar Singh,§ Uttam Gupta,‡ Ranjeet Singh, ‡ Ram Adhar Singh, § and Partha Pratim Manna*, ‡ ‡

Immunobiology Laboratory, Department of Zoology, Institute of Science, and §Department of

Chemistry, Center of Advanced Study, Institute of Science Banaras Hindu University, Varanasi 221005, India. ⊥Department

of Zoology, The University of Burdwan, Bardhaman 713104, India

||

CSIR-Central Salts and Marine Chemicals Research Institute, Bhavnagar, Gujarat. India

S Supporting Information

ACS Paragon Plus Environment

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ABSTRACT ATP is an important transmitter that mediates various biological effects via purinergic receptors (P2-receptors) in cancer. We investigated the anti-tumor activity of ATP-decorated and doxorubicin-loaded mesoporous silica with bio-mineralization of calcium carbonate against a highly aggressive and metastatic murine lymphoma called Dalton’s lymphoma. Our results suggest that this nanocomposite has unique effects with respect to the morphology and properties of calcium carbonate on the surface of the nanoparticle. Doxorubicin in the nanoparticles was prevented from quick release via the interactions of the phosphate group present on ATP and calcium carbonate. This construct is significantly tumoricidal against parental and doxorubicinresistant Dalton’s lymphoma cells and is thus a promising candidate for applications in drug delivery. The composite nanomaterial has excellent biocompatibility with higher uptake and acts via the participation of the purinergic receptor P2X7. The nanocomposite induces significantly higher apoptosis in tumor cells compared with doxorubicin alone. Treatment of Dalton’s lymphoma-bearing mice with the construct significantly reduces tumor burden, in addition to augmenting the lifespan of tumor-bearing mice as demonstrated by a sustained healthy life of the animals and improved histopathological parameters. KEYWORDS: Mesoporous silica, ATP, Doxorubicin, Lymphoma, Biomineralization, P2X7, Apoptosis.


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INTRODUCTION Mesoporous silica nanoparticles (MSNP) perform as a vivid contender for an efficient delivery system and present excellent biocompatibility1, easy functional group employment2, storage capability, and a large surface area and pore size3. Diverse nanomaterial-based delivery platforms have been designed that are controlled by the tumor microenvironment and intracellular signals consisting of the pH4, enzymes5, redox potential6, ATP7 and ROS8. Among them, the ATP-responsive effective drug delivery system draws attention owing to the elevated ATP levels in cancer9. In view of the apparent closeness between ATP and ATP aptamer with respect to binding potential, ATP aptamers were mobilized as a gatekeeper to prevent premature release of the cargo10. Furthermore, identical interactions were employed in the DNA-GO (Graphene Oxide) hybrid nanomaterial to achieve ATP-mediated drug release11. In an additional scheme, the competitive binding between ATP ribose sugar and phenyl boronate completed the release of siRNA from phenylboronate-modified polyion complex (PIC) micelles12. In addition to a role as a drug-liberating operative, other biological aspects of ATP as an antineoplastic agent13 have not been explored in a mesoporous silica-based delivery system. In the past, numerous works have examined the cause of ATP binding to P2X7 purinoceptors, which hold notable importance due to their overexpression in diverse cancer cell types like leukemia, hepatoma and lung carcinoma14-16. These P2X7 purinoceptors are family members of the nonspecific ion channels that facilitate ion transport. ATP promotes the inclusion of biomolecules/particles via membrane permeabilization, which triggers biological responses (membrane swelling, reduced cell proliferation, invasion and apoptosis), suggesting their therapeutic potential in the treatment of cancer via P2 receptors17. This information was further corroborated by Yibo Zhang, who reported an enhancement of cellular uptake plus apoptosis in


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HepG2 cells when the selenium nanoparticles were furnished with ATP18. Therefore, the adjunction of ATP over the doxorubicin (DOX)-loaded MSNP could be a feasible approach to achieve active targeting and a symbiotic apoptotic mechanism. However, this design has some pitfalls, such as hasty drug release from the porous structure and instability of the externally bound ATP, which is hydrolyzed to other metabolic products like ADP, AMP and adenosine. In addition to excellent biocompatibility plus biodegradability, CaCO3-textured nanoparticles have versatile applications in biomedical fields such as gene and drug delivery19, bone repair and tissue engineering20. CaCO3 is stable at pH 7.4 and breaks into Ca2+ and CO32- in acidic environments (pH 5-6)21. Calcium carbonate-decorated nanoparticles can be synthesized with the inclusion of ATP at room temperature. Recently, Chao Qi et al. prepared ACC/ACP (amorphous calcium carbonate/amorphous calcium phosphate) composite nanospheres in which ATP acts as a stabilizer22. Similarly, fructose 1,6 bisphosphate, which contains two phosphate groups, can provide an organic provenance to synthesize calcium phosphate nanostructure materials23. Hence, the ATP-allocated mesoporous silica contributes phosphorous sources for mineralization of CaCO3 over the surface. The mineralization with CaCO3 performs two crucial functions: first, it prevents drug release by sealing the pores; and second, it protects ATP from hydrolysis in the bloodstream. In this study, the potent role of ATP as a potential biomolecule against tumor regression was investigated by using it to decorate the surface of MSNP. Doxorubicin was the drug of choice owing to its successful application for treatment against a wide variety of cancers. The drug was loaded inside the ATP-coupled MSNP, and subsequently, the pores were closed via molecular interactions between ATP and CaCO3. The CaCO3 coating prevented impetuous drug release from MSNP and offered stability to ATP during circulation. Upon reaching the tumor site, the


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acidic environment induced the CaCO3 to leach out into the surrounding medium, causing the exposed ATP to bind to P2X7 purinoceptors present on cancer cells. This novel formulation demonstrated significant tumoricidal activity against Dalton’s lymphoma (DL) and doxorubicinresistant variants of DL cells (DLR)24 with respect to the large-scale arrest of growth and apoptosis. This novel formulation resulted in dramatically higher cellular uptake and a significant apoptotic response in the tumor cells. Therapy with doxorubicin-loaded and ATPcoupled CaCO3-coated MSNP significantly reduced the tumor burden in DL tumor-bearing mice, accompanied by increased survival and the reappearance of normal histo-pathological parameters in treated animals. RESULTS AND DISCUSSION The current study attempted to utilize the collective actions of doxorubicin and ATP as promising candidates for cancer therapy supported by a silica-based delivery system. ATP inherits the antitumor effect and thus functions as a pro-drug. Certain clinical trials using higher levels of intravenous ATP have shown boosted survival rates in patients with pre-terminal cancer. ATP serves as a modulator ligand that binds to P2 purinergic receptors. While bound to these receptors, ATP initiates biological signals, which govern the differentiation and viability of the cell. These P2 purinergic receptors are ATP-responsive ion channels that instigate membrane permeabilization. This non-selective pore opening prompts many biological effects, including membrane blebbing, necrosis, cytokine release and apoptosis via caspase activation25. These purinergic receptors are overexpressed in diverse types of cancer, including ductal adenocarcinoma of the pancreas, breast cancer, and stellate cells, among others. Among the candidates, P2X7 receptor has drawn the attention of many investigators owing to its significance in cell proliferation, migration, invasion and apoptosis. AZ10606120, a potent


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allosteric inhibitor of P2X7 receptor, and high extracellular ATP (0.1–1.0 mM) have shown auspicious outcomes in suppressing tumor cell growth and intrusion26. Moreover, exogenous ATP sensitizes the P2X7 receptor to lead to caspase activation, reduced bromodeoxy uridine (BrdU) embodiment and Annexin V staining. Furthermore, the permeabilization ability allows the uptake of various nanoparticles/biomolecules by cells through the P2X7 receptor. Thus, the cell-penetrating and anti-neoplastic activities of ATP will be advantageous to assimilate in silica supported nanosystems. However, the use of hydrolyzed forms like AMP, ADP or adenosine results in poor outcomes in repressing tumor growth. Consequently, the functions of MSNPcoupled ATP (MSNP-ATP) can be prevented due to the catabolic activity of blood constituents during circulation, which may electrolyze ATP. Furthermore, in many reports, extracellular ATP is considered toxic and has a prominent pro-inflammatory role27. Therefore, to favor ATP stability, restrict DOX drainage and tune the ATP effect towards tumor territories, phosphatederived ATP was employed for the biomineralization with CaCO3. The formed nanosystem (MSNP-ATP-DOX-CaCO3) displayed permanence at physiological pH, while the association between calcium carbonate and phosphate became feeble in the tumor microenvironment where low pH triggers its dissociation from the silica-supported nanosystem. The unmasked ATP then binds to the P2X7 receptor and gives rise to escalated cell-mediated uptake and augments the calcium current28 into the cytoplasm, premiering the accumulation of drug and concurrently disrupting homeostasis. This unique drug delivery system consists of a pH-responsive tumor targeting strategy based on the programmed packing of ATP and CaCO3 molecules over the silica nanoparticles. This mechanistic action shows similarities to the work of Zhen Zou in which MSNP-functionalized folic acid was coated with gelatin molecules. The gelatin molecules were degraded by MMP-2-specific enzymes in the tumor microenvironment, exposing the folic acid to


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bind folate receptor on the cancer cells29. Thus, this new delivery system approach emphasizes the functions of ATP, which together with doxorubicin lead to remarkable cancer cell cessation. A schematic of the synthesis of the CaCO3-capped silica nanoparticles is represented in Scheme 1.

Scheme 1: Schematic of the synthesis of ATP-coupled and doxorubicin-loaded MSNP coated with calcium carbonate. The as-synthesized nanoparticles were studied by transmission electron microscopy (TEM) and showed a nearly spherical shape (Figure S1A) with an average diameter of ~120 nm due to the hexagonal pores for drug loading. The typical honeycomb structure and consequent coating with CaCO3 is presented in Figure S1B and C. A scanning electron microscopy (SEM) image of MSNP-ATP-CaCO3 is presented in Figure S2A, revealing the successful biomineralization of CaCO3 over the surface. Furthermore, the size of CaCO3 was within the 4-10-nm range (Figure S2B), which was sufficient for successful closing of the pores. The energy-dispersive X-ray spectroscopy (EDX) analysis was employed for the elemental analysis of the above-mentioned


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formulations. The EDX elemental mapping of the MSNP-ATP-CaCO3 nanoparticles showed a significant percentage (7%) of calcium over the silica surface (Figure S3). The EDX spectrum of MSNP-ATP and MSNP-ATP-CaCO3 is presented in Figure S4A & B. To systematically communicate the successful annexation steps over the mesoporous silica, various techniques were used to characterize the samples. Brunauer−Emmett−Teller (BET) measurements were carried out to characterize the organization of the MSNP following surface modification. All of the formulations were subjected to the BJH desorption branch of the isotherm for calculating the pore size distribution. BET measurement showed that MSNP displayed a type-IV isotherm curve, forming a hysteresis loop with a specific surface area of 916.43m2/g (Figure 1A) and BJH pore size of 2.6 nm (Figure 1B).

Figure 1. BET nitrogen adsorption/desorption isotherms (A) and BJH pore size distribution of MSNP, MSNP-NH2, MSNP-ATP and MSNP-ATP-CaCO3 nanoparticles (B). A, B, C, and D in Figure 1A represent Bare MSNP, MSNP-NH2, MSNP-ATP and MSNPATP-CaCO3, respectively.


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The attachment of the ATP molecules over the surface of MSNP-COOH resulted in a decline in the surface area to 365.463 m2/g with partial closure of the pores to 2 nm. The molecular size of the doxorubicin was determined using PyMOL Software and was found to be approximately 1.46 nm (Figure S5). Although the pore size of MSNP-ATP was decreased to 2.0 nm, it was sufficient for loading the DOX molecules. Later, when the calcium carbonate was bio-deposited over MSNP-ATP, the surface area was reduced to 129.21 m2/g with no prominent pore, revealing successful sealing of the pores. The additional structural parameters of various functionalized nanoparticles are presented in Table S1. In addition to the nitrogen adsorption/desorption isotherm, wide angle XRD was consigned to monitor the modification over the MSNP. The interaction between biomolecule (ATP)-bound MSNP and the biomineral (CaCO3) was annotated by the wide angle XRD pattern in which the nanocomposite material typifies a nanocrystalline structure (Figure 2A). The observed additional peaks at 2θ=29.30, 35.23, 39.26, 43.35, 47.27, 48.52 and 56.67 closely resembled the calcite structure of the calcium carbonate. We also determined the CaCO3 size from Scherrer’s equation, which provided a value of approximately 12 nm, similar to the values observed in the SEM image. The XRD patterns of MSNP-COOH and MSNP-ATP displayed a broad peak at 2θ in between 20-30 encompassing the amorphous nature of silica nanoparticles. .


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Figure 2 XRD pattern of MSNP-COOH, MSNP-ATP & MSNP-ATP-CaCO3 (A). FTIR spectra of MSNP-NH2, MSNP-COOH, MSNP-ATP and MSNP-ATP-CaCO3 nanoparticles (B) and TGA curves of various functionalized MSNP (C). The Fourier-transform infrared spectroscopy (FTIR) spectra of MSNP-NH2, MSNP-COOH, MSNP-ATP and MSNP-ATP-CaCO3 are shown in Figure 2B. The composed silica nanoparticles displayed specific Si-O-Si and Si-O-related peaks at 1080 cm-1, 800 cm-1 and 464 cm-1, while the broad absorption band at 3450 cm-1 was attributed to stretching of the Si-OH group. The amine functionalized silica nanoparticles were established by a specific peak at 1563 cm-1 ascribed to N–H bending vibrations. Subsequently, the amine functionalized silica


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nanoparticles were carboxylated with succinic anhydride. The composed MSNP-COOH was indicated by a distinct peak at 1720 cm-1 for C=O stretching vibrations in the infrared (IR) spectra. The carboxylic groups over MSNP were stimulated through EDC/NHS to associate with the amine groups in ATP. The ATP-decorated MSNP were established by ultraviolet (UV) absorption at 260 nm (Figure S6) and definite IR peaks at 1238 cm-1 and 1708 cm-1 on account of the stretching vibrations of the PO32- group and the C=N bond in the purine ring, respectively. The CaCO3 coating over MSNP-ATP was exhibited by a distinctive absorbance peak of CO32- at 1428 cm-1. The thermal stability of samples was interpreted by thermogravimetric analysis (TGA) to further endorse the functionalization steps. The TGA curves of MSNP-NH2, MSNPCOOH, MSNP-ATP and MSNP-ATP-CaCO3 are presented in Figure 2C. All of the curves showed weight loss below 100°C that can be ascribed to the absorbed water that is widely present in the amorphous phase. The TGA curve of MSNP-ATP represents weight loss between 200 and 500°C, which was associated with the decomposition of ATP molecules. The biomineralization of CaCO3 over MSNP-ATP formed an inorganic corona over the surface of the silica nanoparticles, which represents marginal weight loss between 200˚C-500˚C. Later, when the temperature exceeded 600°C, weight loss was mainly assigned to the degradation of CaCO3 in MSNP-ATP-CaCO3. Dynamic light scattering (DLS) studies have examined the hydrodynamic size of nanoparticles like MSNP-NH2 and MSNP-ATP-CaCO3 with a mean diameter of ~140 nm and ~190 nm, respectively, in PBS (10 mM, pH 7.4) (Figure S7A). The amplification of the particle diameter of MSNP-ATP-CaCO3 can be explained by various conjugation steps over the surface of MSNP that augment its size. Similarly, the zeta potential changes with each modification step suggest that varying moieties were conjugated to the MSNP surface. The amino co-condensed MSNP possessed a zeta potential of +12.3 ±0.81 mV, which switched to


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−27.5 ±1.0 mV after succinylation because of the derivatized carboxyl acid groups (Figure S7B). Following the conjugation of ATP, the zeta potential declined to -35.6 ±0.97 mV due to the presence of phosphate groups. Following subsequent conjugation to calcium carbonate, the zeta potential of the formed MSNP-ATP-CaCO3 nanoparticles shifted to -15.8 ±1.2 mV, consistent with previously reported values22. We also determined the ATP content, which was conjugated to MSNP-COOH through its strong UV absorption potential. MSNP-ATP was subjected to UV absorption, and the observed value was then subtracted from the baseline value of MSNP-COOH. The obtained absorbance value was compared with the standard curve of ATP to determine its concentration. Our data revealed that approximately 77.5 µg of ATP was linked per milligram of silica nanoparticles. Furthermore, we also demonstrated that approximately 82.7 µg of the ATP was chemisorbed over the silica nanoparticles, as judged using an ATP determination kit (Abcam). We performed gate opening of the construct by doxorubicin release at three different pH values (7.4, 6.2 and 5.4). The purpose of this experiment was to demonstrate the release of doxorubicin at increasingly acidic pH values from 7.4 to 5.4. The results clearly demonstrated that the melting down of CaCO3 in acidic pH compartments led to significantly greater release of doxorubicin at pH 5.4 compared with pH 6.2 in a time-dependent manner for an extended period of 72 hours. pH 7.4 clearly stabilized the construct with negligible release (Figure S8A). In contrast, cargo (DOX) released from MSNP-ATP only was significantly faster compared with CaCO3-wrapped MSNP-ATP-DOX, suggesting the rapid dissolution of the cargo in unprotected MSNP-ATP (Figure S8B). The release data clearly demonstrate that bio-mineralization with CaCO3 offers significant advantages compared with MSNP-ATP-DOX, suggesting its suitability for the tumor microenvironment.


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We assessed the tumoricidal activity of doxorubicin-loaded MSNP-ATP-DOX-CaCO3 against an aggressive and highly metastatic murine lymphoma called Dalton’s lymphoma (DL). We also tested the efficacy of the construct system against doxorubicin-resistant DL cells called DLR. DLR cells were engineered in vivo in mouse peritoneum subjected to treatment with doxorubicin. DLR cells were tolerant to doxorubicin with significantly higher IC50 values than DL (Figure S9). Our data suggested that DOX-loaded MSNP-ATP-DOX-CaCO3 significantly retarded cellular proliferation in the presence of varying concentrations of the constructs. Assessment of tumor cell percent inhibition at 24 hours and 48 hours in the presence of increasing concentrations of the compound including MSNP-ATP-CaCO3 was studied (Figure 3A-D). In contrast to free DOX or ATP, MSNP-ATP-DOX-CaCO3 was significantly more tumoricidal against DL and DLR cells. Incubation of DL cells with MSNP-ATP-DOX-CaCO3 for 48 hours caused an extensive reduction of proliferation (Figure 3). We then compared the percent proliferation of varying numbers (5×103 and 20×103) of DL and DLR cells in the presence of a fixed concentration (25 µg/mL) of the indicated formulations. Compared with DLR cells, DL cells were effectively killed by the formulation even with a higher target load (Figure S10). The percent proliferation of 20×103 DL cells was reduced by 40% in the presence of MSNP-ATP-DOX-CaCO3 (p

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