Regular Article
Nanogold-core multifunctional dendrimer for pulsatile chemo-, photothermal- and photodynamic- therapy of rheumatoid arthritis

https://doi.org/10.1016/j.jcis.2019.02.073Get rights and content

Abstract

This investigation reports a novel nanoGold-core multifunctional dendrimer for pulsatile chemo-, photothermal- and photodynamic- therapy of rheumatoid arthritis (RA). Architecturally, the nanocomposites comprised of a nanoGold (Au) at the focal whose surface is functionalized by hydroxy-terminated thiolated-dendrons following Au-thiol bond formation to produce nanoGold-core multifunctional dendrimer (Au-DEN). The surface hydroxyl groups of Au-DEN were then conjugated with methotrexate (MTX; a disease-modifying first line anti-rheumatic drug; DMARD; 74.29 ± 0.48% loading) to form Au-DEN-MTX-NPs (Particle size: 100.15 ± 28.36 nm; poly dispersibility index, PDI: 0.39 ± 0.02; surface zeta potential, ζ: −22.45 ± 1.06 mV). MTX was strategically selected to serve as an anti-rheumatic DMARD as well as a targeting ligand to attain selective localization of the formulation in arthritic tissue via folate receptors upregulated on arthritic tissues. The docking study was performed to confirm the viable binding efficiency of MTX towards β-folate receptors that are overexpressed on arthritic tissues taking folic acid as a reference standard. The IR780, a NIR active bioactive was also loaded in Au-DEN-MTX NPs to offer photothermal benefit upon irradiation with NIR laser (wavelength: 808 nm). The hypothesis was tested by elucidation of in vitro drug release profile, photothermal activity, cellular uptake (Fluorescence and confocal laser scanning microscopy; CLSM), cell viability assay (MTT protocol) and Intracellular reactive oxygen species (ROS) generation in mouse macrophage RAW264.7 cells and Lipopolysaccharide (LPS) activated RAW264.7 cells. Furthermore, the hemolytic toxicity and stability studies were also investigated to determine the blood compatibility as well as ideal storage condition of NPs. The outcome of this investigations presents developed multifunctional targeted NPs to be potential therapeutics for the improved treatment of RA. The approach can also be applied to other clinical interventions involving countering inflammatory conditions.

Introduction

Gold (Au) is one of the first metal to have been discovered in the history of human civilization; that has the history of at least several thousand years for its application spans in various therapeutic and non-therapeutic fields [1]. In countries like India and Nepal, Au ashes (locally known as Bhasmas) have been described in the 5000-year-old Ayurvedic system native to the Indian subcontinent for healing diseases like rheumatoid arthritis (RA) [2]. Last few decades have witnessed an extensive use of Au based nanocomposites such as Au-nanoshell, Au-nanorods, Au-nanoseeds, Au-nanoplates or Au-nanoparticles for photothermal therapy owing to their capability to absorb near infra-red (NIR) light and generate heat responsible for localized cell ablation or to attain cellular apoptosis [3], [4]. Recent studies showed that multifunctional Au-nanocomposites to be proficient in simultaneously delivering both heat and drug to the desired site of action [5], [6].

RA is a chronic autoimmune disorder of multiple joints which symmetrically affects the whole body and can progress to systemic complications, disability, early death and socioeconomic costs [7]. It mainly affects the joints, tendons, connective tissue, fibrous tissue, and muscle. As the disease advances, there is evidence of gradual destruction of the synovial capsule, cartilage lining, pannus formation, angiogenesis, bone erosion, stiffness in joints and unbearable pain [8].

Methotrexate (MTX) is folate analog that has its physicochemical and structural properties substantially similar to those of folate. It acts as an inhibitor of dihydrofolate reductase and thereby interferes with DNA synthesis by reducing the purine and pyrimidine supply to the rapidly dividing cells. This antiproliferative activity of MTX is accomplished with high-dose regimens and is the basis for its application for clinical intervention of cancer. Interestingly, in low dose, MTX is well-established as a disease modifying anti-rheumatic drug (DMARD) to mediate immunomodulatory and anti-inflammatory effect [9]. Its anti-inflammatory effect is exerted by inducing extracellular adenosine predominantly through G-protein-coupled adenosine (type A2a also known as ADORA2A) receptors to inhibit the expression of interleukins (ILs), tumor necrosis factor-α (TNF-α) in monocytic cells and thereby acts to inhibit cell-mediated immunity to provide relief in the symptoms of RA [10].

Lee et al. synthesized RGD anchored Au-nanoshell for targeted delivery of MTX for RA therapy. When irradiated with near infrared irradiation (NIR) laser, the Au-nanoshell generated the heat along with simultaneous and sustained release of MTX in inflamed arthritic area of collagen induced arthritic mice. It was observed that the NIR mediated photothermal approach increased the sensitivity and therapeutic efficacy of MTX as compared to the traditional MTX therapy [11]. Costa Lima and co-workers also investigated Au-based stealth nanospheres of MTX for RA therapy. The authors observed a pH dependent in vitro release behaviour of MTX from Au-based stealth nanospheres following photothermal activation of its Au component. A significant reduction in monocytes and proinflammatory cytokines (also known as inflammation mediators) including interlukein-1β, interlukein-6 and tumor necrosis factor-α in in vitro studies [12].

It has been widely reported that β-folate receptors are profoundly overexpressed in the inflamed arthritic tissues. Overexpressed. Keeping this fact in view, Thomas et al. investigated the anti-arthritic potential of MTX delivered in its conjugated form with folate anchored Polyamidoamine (FA-PAMAM-MTX). The developed FA-PAMAM-MTX showed selective internalization in the inflamed RA tissues in receptor specific manner by utilizing the β-folate receptors that overexpressed. The MTX delivered by this targeted approach produced a potent anti-inflammatory effect with marked reduction in arthritis-related events such as joint inflammations, paw volume, cartilage damage, redness, bone resorption and change in body weight [13]. In another investigation, Benchaala et al. also functionalized PAMAM dendrimer with MTX to investigate the targeting potential of peripherally conjugated MTX. The authors found observed four-fold higher localization of MTX conjugated PAMAM at the inflamed arthritic area as compared to the unmodified PAMAM dendrimer loaded physically with MTX. The authors ascribed the higher and selective localization of MTX conjugated PAMAM dendrimer to be because of selective receptor mediated uptake via folate receptors overexpressed in the inflamed arthritic tissues [14].

This manuscript reports an innovative and novel approach using nanoGold-core multifunctional dendrimer for pulsatile chemo-, photothermal- and photodynamic- therapy of RA. Architecturally, the nanocomposites comprised of a nanoGold (Au NP) at the focal whose surface is functionalized by hydroxy-terminated thiolated-dendrons following Au-thiol bond formation to produce nanoGold-core multifunctional dendrimer (Au-DEN). The surface hydroxyl groups of Au-DEN are then conjugated with methotrexate (a DMARD) to form Au-DEN-MTX-NPs. MTX was strategically selected to serve as DMARD as well as a targeting ligand to attain selective localization of the formulation in arthritic tissue via folate receptors upregulated on arthritic tissues to mediate an improved treatment intervention of RA. The docking study was employed to confirm the viable receptor binding efficiency of MTX that of folic acid towards β-folate receptors that are overexpressed on ailing RA tissues. The IR780 (a NIR active bioactive) was also loaded in the loading crevices of Au-DEN-MTX with an aim to offer photothermal benefit upon irradiation with skin permeable NIR laser of 808 nm wavelength.

The core objective of this investigation was to develop a multifunctional NPs therapeutics platform that can help to simultaneously attain a combined effect of MTX, IR780, and Au while availing synergistic increment in their respective therapeutic efficacies with significantly lowered side effects associated with their individual therapies by mediating β-folate receptor-mediated targeted RA therapy. The hypothesis was tested by elucidation of in vitro drug release study, photothermal activity, cellular uptake assay (fluorescence and confocal laser scanning microscopy; CLSM), cell viability assay (MTT protocol) and Intracellular reactive oxygen species (ROS) generation was studies for various formulations under investigation in mouse macrophage RAW264.7 cells and Lipopolysaccharide (LPS) activated RAW264.7 cells. Furthermore, the hemolytic toxicity and stability studies were also studied to determine the blood compatibility as well as ideal storage condition for the storage of developed NPs. It is anticipated that this approach can also be applied to other clinical interventions involving countering inflammatory conditions.

Section snippets

Materials

MTX was received as a kind gift from Amneal Pharmaceuticals, Ahmedabad, India. Gold chloride tri-hydrate, (1-(3-Dimethylaminopropyl-3-ethylcarbodiimide, hydrochloride) (EDC), 4-dimethylamino pyridine (DMAP), Polyester bis-MPA DEN, 16-OH, 1-SH-4th generation (Polymer factory, Sweden), Trehalose di-hydrate, TNF-alpha, IL-6 and IL-1B were purchased from Sigma Aldrich St. Louis, USA. Acetonitrile HPLC Grade, Glacial acetic acid, Sodium hydroxide, Methanol SQ grade procured from Fisher Scientific,

Synthesis and characterization of Au-NP and Au-DEN-NP

This study reports a novel Au-NP-core multifunctional dendrimer approach for pulsatile chemo-, photothermal- and photodynamic- therapy of rheumatoid arthritis. For the preparation of novel Au-NP-core multifunctional dendrimer NPs, a step-by-step synthesis methodology was adopted as presented in Fig. 1. Architecturally, the nanocomposites comprised of an AuNP at the focal point. The Au-NPs were synthesized using the citrate reduction turkevich method and purified using centrifugation technique

Conclusion

It can be concluded that a therapeutic intervention for RA therapy that involves NIR laser responsive Au-DEN-MTX-IR780 NPs has been successfully developed and preclinically tested for targeted chemo-photothermal treatment of RA in this investigation. The strategy comprises of MTX, a DMARDs drug combined with NIR laser generated photothermal heat that plays a vital role in ROS generation to provide a synergistic opportunity to effectively treat RA. The outcome of this investigation conclusively

Disclosures

There is no conflict of interest and disclosures associated with the manuscript.

Acknowledgment

RKT would like to acknowledge Science and Engineering Research Board (Statutory Body Established Through an Act of Parliament: SERB Act 2008), Department of Science and Technology, Government of India for the grant (Grant #ECR/2016/001964) for research work on drug and gene delivery. RKT and RM would also like to thank DST-SERB for N-PDF funding (PDF/2016/003329) for the development of targeted cancer therapeutics in Dr. Tekade’s Laboratory. The research team is thankful to Dr. Alok Jain, Mr.

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