Bisphosphonate-conjugated silica nanoparticle internalization and localization patterns in osteoclast and osteoblast precursor cells Open Access

Abstract

In the past decade, Nanotechnology has emerged as an important novel strategy for

drug delivery and therapeutic intervention. Silica nanoparticles especially have proven

revolutionary in the realm of bone biology. Their ability to be readily internalized in bone cells

and general biocompatibility open up opportunities for new bone disease treatments, such as

osteoporosis. Bioactive, OH-terminated silica nanoparticles are particularly promising due to

their ability to affect the differentiation of osteoblasts and osteoclasts in vitro. As opposed to

current osteoporosis drugs which only suppress osteoclast activity, these nanoparticles are

unique in their ability to inhibit osteoclast differentiation, the cells responsible for bone

resorption, and stimulate osteoblast differentiation, the cells responsible for bone formation.

Additionally, the nanoparticles have been shown to increase bone density in mice in vivo.

However, to improve targeting to the bone, and thereby increase efficacy, a modified

nanoparticle with alendronate, a bisphosphonate used for osteoporosis, on the surface was

designed. In this study, we show that OH-terminated silica nanoparticles and bisphosphonate-

conjugated nanoparticles exhibit similar internalization patterns with respect to time and

concentration in both pre-osteoclast RAW 264.7 and pre-osteoblast MC3T3-E1 cell lines.

Endocytosis studies also indicate shared internalization pathways between the two particles

and colocalization assays identify shared localization patterns. In addition, XTT assays

demonstrate no decrease in viability for cells treated with bisphosphonate-conjugated silica

nanoparticles. These studies pave the way for future functional assays and provides the first

step in elucidating the characteristics and mechanistic behavior of the newly developed

bisphosphonate-conjugated nanoparticle. As opposed to merely slowing the degradation of

bone, our nanoparticle would have the ability to restore lost bone density. Combined with

improved targeting to the bone, this nanoparticle has the potential to be a highly effective new

treatment for osteoporosis. 

Table of Contents

Introduction ........................................................................................................................ 1

Materials and methods ......................................................................................................... 2

Silica nanoparticle synthesis................................................................................................. 2

Characterization of silica nanoparticles.................................................................................. 2

Cell culture and reagents....................................................................................................... 2

Dose-response internalization assays .................................................................................... 2

Time-course internalization assays ....................................................................................... 3

Endocytosis assays............................................................................................................... 3

Colocalization assays ........................................................................................................... 4

XTT assays........................................................................................................................... 4

Fluorescence microscopy imaging ......................................................................................... 4

Statistical analysis................................................................................................................ 4

Results ................................................................................................................................ 4

Silica nanoparticle characterization ...................................................................................... 4

Dose-dependent internalization of OH-terminated and bisphosphonate-

conjugated silica nanoparticles ............................................................................................. 5

Time-dependent internalization of OH-terminated and bisphosphonate-

conjugated silica nanoparticles ............................................................................................. 5

Endocytosis assay of bisphosphonate-conjugated silica nanoparticles ..................................... 6

Colocalization assay of bisphosphonate-conjugated silica nanoparticles .................................. 7

XTT assay of bisphosphonate-conjugated silica nanoparticles................................................. 7

Discussion......................................................................................................................... 17

Conclusion ........................................................................................................................ 19

References ......................................................................................................................... 20 

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, General Nanotechnology Biology, Physiology
Keyword	Silica Nanoparticle Osteoclast Osteoblast Osteoporosis Nanomedicine
Committee Chair / Thesis Advisor	George Beck, Emory University
Committee Members	Jamie Arnst, Emory University Eladio Abreu, Emory University M Neale Weitzmann, Emory University

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