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Protease Responsive Targeted Delivery of Doxorubicin from BilirubinBSA Capped Mesoporous Silica Nanoparticles against Colon Cancer Prateek Srivastava, Sumit Kumar Hira, Divesh N Narayan Srivastava, Uttam Gupta, Pradip Sen, Ram Adhar Singh, and Partha Pratim Manna ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00635 • Publication Date (Web): 05 Oct 2017 Downloaded from http://pubs.acs.org on October 7, 2017

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ACS Biomaterials Science & Engineering

Protease Responsive Targeted Delivery of Doxorubicin

from

Bilirubin-BSA

Capped

Mesoporous Silica Nanoparticles against Colon Cancer Prateek Srivastava

†, ⊥, ∥ ,

Sumit Kumar Hira‡, ∥, Divesh Narayan Srivastava§, Uttam Gupta†,

Pradip Sen#, Ram Adhar Singh⊥ and Partha Pratim Mannaa* †.

†

Immunobiology Laboratory, Department of Zoology, Institute of Science, Banaras Hindu

University, Varanasi – 221005, India.

⊥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.

#

Institute of Microbial Technology, Chandigarh, India

KEYWORDS: Mesoporous silica, Bilirubin, Doxorubicin, Colon cancer, Apoptosis.

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ABSTRACT

Bilirubin is regarded as a toxic waste, produced from heme degradation and also acts as a potentially important antioxidant. Bilirubin causes arrest in cell cycle and lead to lesser occurrence of malignancies in individuals, having normal or slender increase in levels of serum bilirubin. Prompted by the dynamic interaction between bilirubin and bovine serum albumin (BSA), bilirubin-BSA complex was explored as a biocompatible cap system for protease responsive delivery device of anti-cancer drug against colon cancer. Bilirubin, conjugated to the amine terminated and doxorubicin loaded mesoporous silica nanoparticles were employed as a novel formulation against colon carcinoma cells. Compared to doxorubicin only, bilirubin in combination with doxorubicin loaded mesoporous silica nanoparticle significantly inhibits tumor cell growth as assessed in MC-38 (murine) and HCT-116 (human) colon cancer cells. Bilirubin-doxorubicin combination potently inhibits proliferation of tumor cells and acted as cytotoxic and pro-apoptotic agent in vitro. Our result demonstrates that this novel cap system could play a precise role in defense against colon cancer by interrupting the pro-cancerogenic survival pathways during carcinogenesis.

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INTRODUCTION

Mesoporous silica nanoparticles (MSNP) represent inorganic support for generating various cap systems which can be orderly regulated via external stimuli. These gated materials provide the controlled movement of active cargos from voids of porous supports. For designing of cap systems, certain polymers1, cyclodextrins2, rotaxanes3, DNA aptamers4, methacrylates5, layer-by-layer6 (LBL) and metal complexes7 were explored to prevent the deliverance of cargo from porous hosts and, there by optimize their controlled release behaviors. Various triggering stimuli that are used for unlocking the pores includes pH8-11, temperature12, light13, esterase activity14, protease cleavage15 and the redox reactions16 fundamentally linked to the various biological systems. Recent developments for making biocompatible capping agents have employed biologically active molecules such as nucleotides17, biotin-avidin complex18, chitosan19, polyglutamic acid20, cellulose21 and collagen16 to block the pores reversibly. Among them, biotin-avidin complex shows strong non-covalent interaction which links a protein with its ligand. Biotin rapidly forms bond with avidin and after its formation it is protected from extreme pH, organic solvents, temperature or any such denaturing agents. Thomas Bein and his colleagues reported that the proteinligand reciprocal action can be employed in blocking the pores in colloidal mesoporous silica nanoparticles18. In several other systems, opening of the pore was regulated by direct cleavage through MMP9 specific linker22 or decrease in protein bonding interactions by proteolytic degradation18. Availing such protein-ligand interaction in covering the pores, we herein, employed Bilirubin-BSA complex as a novel capping agent in protease responsive drug release from MSNP.

Bilirubin is an endogenous yellow colored compound consisting of open chain four pyrrolelike rings, derived from various heme proteins like hemoglobin, cytochrome P-450, 3



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myoglobin, etc.23 Due to poor water solubility, bilirubin is represented in the conjugated form with albumin and ferried in the plasma24. The molecular interaction of bilirubin with BSA can be studied by circular dichroism (CD) and spectrofluorimetry25. Three binding sites in bilirubin identified as subdomain IB, IIA and IIIA with different affinities towards BSA and are instrumental for its strong non-covalent interactions26. Bilirubin alone is optically inactive and demonstrates extrinsically enhanced cotton effect in the region of the bilirubin absorption maximum when complexed with BSA27.

Beside these studies, bilirubin also play significant role in arresting the proliferation of cancer cells. Bilirubin induces oxidative stress which results in upregulating the PTEN (Phosphatase and tensin homolog) expression through APE1/Ref-1 signaling pathways28. Tumor formation is also accompanied by mutations and events like chromosomal deletion which inactivates PTEN enzymatic activity that trigger uncontrolled proliferation and subsequent reduction in cell death29. PTEN referred as a key tumor suppressor protein, has the tyrosine phosphatase activity besides playing the role of a counter regulatory mechanism to phosphoinositide 3kinase/AKT mitogenic signaling pathway30. PTEN acts via its lipid phosphatase activity and suppress phosphoinositide 3-kinase (PI3K)–AKT–mammalian target of rapamycin (mTOR) pathways, and actively perform a significantly dominant role in cancer.

In the current study, Bilirubin-BSA complex was explored as a biocompatible cap system with the protease responsive pore opening, for treatment against colon carcinoma. Bilirubin was conjugated to the amine terminated silica nanoparticles. Doxorubicin (DOX) was loaded inside MSNP and the pores were sealed via molecular interactions, likely occur between the BSA and Bilirubin. This formulation represents a collusive nature in which the cap system prevents the premature drug release from gated material besides the additional role for delivering antitumor punch by increasing apoptosis in tumor cells. The DOX loaded MSNP 4 ACS Paragon Plus Environment

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shows good colloidal stability with impressive cancer cell death. The formulation significantly affects the human (HCT-116) and murine (MC-38) neoplastic cells derived from colon carcinoma by inhibiting the amplification of cell number and inducing apoptosis.

RESULTS AND DISCUSSION

Detailed characterization of MSNP

For designing an effective drug delivery system, various capping agents were constructed to gate biological samples/drugs which could finely tuned to the external or internal stimulus of the surroundings. Advances in the fabrication of biocompatible capping agents in biomedicine have provided efficacy and safety with new therapies. In lieu of protein ligand interactions, biotin-avidin complex were fabricated as a new capping agent over MSNP surface for protease responsive guest release. Recently, gossypol-capped and mitoxantroneloaded MSNP showed the possibility to achieve new advanced predesigned function where gossypol, an anticancer drug was linked through the boronate ester bond to the mitoxantrone loaded MSNP31. Gossypol represented as a capping device which demonstrates the cooperative cytotoxicity towards the cancer cells. However, owing to its no approval status, chemotherapy with gossypol as a delivery system concerns with some drawbacks. In this study, our strategy was to design and develop a cap system which can fulfill the criteria to prevent the drug leakage from the porous support and own the antitumor activity and concurrently biocompatible to prevent any biological responses during circulation. Herein, we address Bilirubin-BSA complex as a novel biocompatible protease responsive cap which have biphasic functions, first to regulate the transport of drug (doxorubicin) from the pores and secondly the physiological support as a cellular antioxidant which enhances the tumor suppression in association with the drug. Bilirubin was regarded as a keystone biomolecule in 5



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designing the cap system which forms strong molecular interactions with BSA and induces antitumor activity by enhancing the PTEN expression28. Considering the normal transport method of bilirubin with BSA in conjugated form in the plasma, the mesoporous silica capped bilirubin-BSA complex showed the biological mimicry to prevent any inflammatory or hemolytic responses.

The Bilirubin-BSA complex over the MSNP forms a reliable cap system besides performing role in protein receptor mediated cellular uptake. One study demonstrates that the BSA corona over the functionalized mesoporous silica nanoparticles has lead to preferable cellular uptake with higher drug biodistrubution in the cancer cells32. Further, there are reports which focus on the cytotoxicity information and biological effects of the cross linkers, utilized over the surface of nanoparticles for bioconjugation33. Herein, we introduce a heme by-product bilirubin as a natural cross linker which inherits brilliant biocompatibility and binding abilities with BSA. The scheme of the protease responsive doxorubicin delivery system is represented in the Figure 1.

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Figure 1. Scheme of synthesis for Bilirubin-BSA cap system for protease mediated drug release.

The formed MSNP were synthesized from the earlier reported methods using sol gel chemistry. TEM represents the spherical texture of the as synthesized MSNP with an average particle of approximately 120 nm in size having honeycomb like pores (Figure 2A). The features in enlarged version of the nanoparticle demonstrate characteristic honey comb design for housing the doxorubicin (Figure 2B).

Figure 2. TEM images of the MSNP. Scale bar is 1 µm in A and 100 nm in B

The Si−O−Si peaks observed in FTIR spectrum at 1080 and 800 cm−1were ascribed as stretching vibrations and the corresponding peak at 459 cm−1was because of bending vibration, both validating the nanoparticle formation (Figure 3A). Amine-functionalization of MSNP was mediated following reaction with APTMS, as evidenced by the occurrence of peak at 1540 cm−1 corresponds to N-H bending vibrations. Bilirubin composed of linear tetrapyrrole dicarboxylic acid which can be evident by specific peaks at 1696 cm-1 and 3267 cm-1 representing carboxylic and the vibrational N-H group (lactam) in the backbone 7



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structure. The covalent tagging of bilirubin with MSNP-NH2 can be demonstrated by decline of the amine group peak, reflecting decrease in number of free amine group content in the IR spectra, which suggest possible molecular interactions transpire between the bilirubin’s carboxylic groups and the amine groups of MSNP. UV-vis spectroscopic and TGA studies further expands the successful formation of bilirubin garnished MSNP. Bilirubin is a chromophore which tends to absorb strongly at 265 nm because of the occurrence of linear pyrrole rings (Figure 3B). A similar type of UV absorption band was achieved when MSNP was conjugated with bilirubin.

Figure 3. (A) FTIR spectra of MSNP-NH2, free bilirubin and MSNP-BR. (B) Represents the UV absorbance graph of MSNP-NH2, free bilirubin and bilirubin attached MSNP.

In TGA analysis, the occurrence in thermal degradation events of various functionalized nanoparticles was evident by the increased weight loss compared to bare MSNP which clearly certify the conjugation steps (Figure S1). Surface modifications over the MSNP can be demonstrated by the change in surface area besides the pore volume. Bare MSNP results a type IV isotherm with a specific BET surface area measurement of 1243 m2 gm-1 and BJH pore size of 3.34 nm (Figure 4). However, the systematic attachment of bilirubin over the 8 ACS Paragon Plus Environment

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surface of nanoparticle leads to decline in the available surface area which becomes 867 m2 gm-1 with the partial closure of pores to 2.92 nm. The molecular interactions between bilirubin attached MSNP and the large BSA protein molecules have the proficiency to reduce the pores in the porous material which results a remarkable decrease in the area of nanoparticle surface to 315 m2 gm-1 and with minimum pore size compared to bare MSNP. This indicates the successful construction of biocompatible cap system. All the structural details of various nanoparticles are given in (Table S1).

Figure 4. Represents the Nitrogen Adsorption desorption isotherm (A) MSNP (a), MSNPBR (b) and MSNP-BR-BSA (c). BJH pore size (B) distributions of the various functionalized nanoparticles as indicated in MSNP, MSNP-BR and MSNP-BR-BSA.

The biological attachment between bilirubin and the BSA was further studied by circular dichroism and fluorescence. Earlier observation indicates that bilirubin is an optically inactive biomolecule, although its conjugation over MSNP results minimal optical rotation close to MSNP-NH2 (Figure 5A). Nevertheless, a strong negative cotton effect was observed when the MSNP-Bilirubin was complexed with BSA. Further the CD spectrum of MSNP bound BSA was moderately dissimilar compared to the CD spectrum of free BSA which can 9



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be explained by the fact that the protein’s secondary structures (α-helix and β-sheets) were engaged in the molecular interactions with the bilirubin attached silica nano system. Similarly, the fluorescent property of bilirubin was studied which results in a low fluorescence, with an emission maximum at about 530 nm when excited at a wavelength of 448 nm27. When bilirubin was complexed with BSA molecule, its fluorescent property was augmented which was assisted by protracted quenching of inherent fluorescence of the protein. An enhanced fluorescent spectrum was obtained when the bilirubin attached MSNP form the molecular association with the BSA molecules through the non-covalent interactions (Figure 5B).

Figure 5. (A) CD spectra of different functionalized nanoparticles as MSNP-NH2, MSNP-BR, MSNP-BR-BSA and the free BSA in PBS (pH 7.4). (B) Emission spectra of MSNP-NH2, MSNP-BR and MSNP-BR-BSA in PBS (pH 7.4) when excited with wavelength at 448 nm.

We also determined the mass of BSA attached over the MSNP-Bilirubin, which was measured by Bradford assay method. Our data reveals, approximately 72.5 µg of BSA was attached per milligram of silica nanoparticles. The amount of BSA attached nonspecifically


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over the amine terminated mesoporous silica was approximately 28 µgmg-1 of nanoparticles, as demonstrated by Bradford method.

DLS of the MSNP-BR and the MSNP-BR-BSA were conducted in PBS (10 mM, pH 7.4) solution. The representative mean particle size for MSNP-BR and the MSNP-BR-BSA was 160 nm and 210 nm respectively (Figure S2 A). To further confirm the conjugation over the surface geometry of MSNP, zeta potential measurement was conducted to validate the conjugation step over the silica nanoparticles in presence of 10mM PBS solution at pH 7.4. The bare MSNP represents specific charge of -27 mv which was on account of the Si-OH group’s presence over the surface; however, after the condensation with the amino propyl group, shifting in zeta potential occur towards the positive and become +6.8 mv. Later, when bilirubin was conjugated through the carboxyl groups, the reduction in zeta potential was observed which reduces to -5.4 mv, indicating the decreased amount of amine group over the structure of nanoparticles (Figure S2 B). Following the molecular interaction between the BSA and the MSNP-BR, the zeta potential further reduces to -12.2 mv. Further, the attachment of BSA over the amine terminated silica was confirmed by the change in the values obtained in zeta potential, which switched from +6.8 mv to -10.8 mv when the protein was reversibly attached. The DLS of the amine capped silica nanoparticles is presented in the Figure S3.

Protease responsive DOX release from MSNP-BR-DOX-BSA

BSA-Bilirubin novel biocompatible complex was formulated for protease-responsive targeted release of DOX in colon cancer cells. Our results demonstrate a time dependent increase in availability of DOX concentration after treatment with trypsin. Trypsin acts on BSA which coats the DOX loaded MSNP and thus release the DOX, suggesting the uniquely designed 11



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efficiency of the formulation for slow and sustained release in cellular environment in presence of protease (Figure 6A).

Earlier studies implied that MSNP have the power to interact with BSA in a non-specific manner during circulation in blood streams34. Thus, we compare the binding abilities of both bilirubin and amine terminated silica with BSA to address the stability issue at physiological pH 7.2 for 48 hours. We conclude that BSA-bilirubin conjugate provides more stability by conferring better encapsulation of the drug within the nanosystem. The amine conjugated BSA couldn’t hold up the doxorubicin to the same extent, implying that the reciprocal actions between BSA and amine terminated silica were not substantially strong to block the pores reversibly for a long duration of time. MSNP-BR-DOX-BSA formulation is significantly stable compared to MSNP-NH2-DOX-BSA at pH7.2 (Figure 6B). Time dependent kinetics study of DOX release reveals that the MSNP-BR-DOX-BSA performs significantly better in retaining the DOX compared to MSNP-NH2-DOX-BSA. This result suggests that bilirubin attachment to DOX loaded MSNP makes the nanoparticle comprehensibly sound and a better device for therapeutic purpose.

We also demonstrated the BSA content effused from the Bilirubin terminated cap system when seeded at the physiological pH 7.2 for prolonged time period. The MSNP-BR-BSA nanoparticles were incubated in PBS (1×) at 4°C and 37°C. The supernatant was collected at specific time points for determining the BSA concentration via Bradford method. We conclude that the BSA content leached form bilirubin terminated cap system at 37°C was approximately 1.20 µg after 120 hours (Figure S4). At 4°C, the construct is highly stable and leached very little BSA (~0.5 µg) even after incubation for 5 days. This result suggests that the bilirubin as a ligand inherits the strong affinity towards BSA which prevents its discharge


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from the particle surface and manifests the stability of the cap system for the extended period of time.

Figure 6 (A) Doxorubicin release kinetics from MSNP-BR-DOX-BSA in presence of trypsin treatment. (B) Comparative assessment of doxorubicin release from MSNPNH2-DOX-BSA and MSNP-BR-DOX-BSA at pH 7.2.

Tumoricidal activity of MSNP-BR-DOX-BSA against colon cancer

For developing a cap system with an antitumor function, we further investigated the performance of both the cap systems to arrest the tumor growth. Here, we establish that bilirubin cap system by virtue of its cogent antioxidant and proto-oncogene regulatory activities, demonstrate better killing of cancer cells. The amine terminated cap system was unable to induce any effect against lower or higher concentrations of tumor cells when compared to bilirubin cap system (Table S2). MSNP-BR-DOX-BSA demonstrates significant tumoricidal properties against murine MC-38 and human HCT-116 colon carcinoma cells. We compared the antitumor activity of MSNP-bilirubin, free DOX, MSNPBR-DOX and MSNP-BR-DOX-BSA against varying numbers (5×103 and 20×103) of target 13



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cells to demonstrate the applicability and efficacy of the formulation following increase in tumor cells mass in advanced stages of cancer. Concentration dependent kinetic study suggests, MSNP-BR-DOX-BSA shows significantly higher anti-proliferative effect against HCT-116 (5×103cells) target cells compared to doxorubicin or bilirubin alone (p

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