Clinical Relevance of Calcium Phosphosilicate Nanoparticles for the Treatment of Human Bone Disease
Open Access
- Author:
- Gigliotti, Christopher
- Graduate Program:
- Bioengineering
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- February 07, 2019
- Committee Members:
- James Hansell Adair, Dissertation Advisor/Co-Advisor
James Hansell Adair, Committee Chair/Co-Chair
Gary Lynn Messing, Committee Member
Daniel J Hayes, Committee Member
Gregory Stephen Lewis, Outside Member - Keywords:
- nanoparticles
drug delivery
bioconjugation
design of experiment
3D printing
metastatic breast cancer - Abstract:
- Nanomedicine is currently being explored with the intent of leveraging advances in material science and clinical technology to overcome the barriers associated with traditional drug delivery. Clinical outcomes for bone disease such as metastasized breast cancer in bone are reliant on early detection and efficacious treatment with limited adverse events. Synthesizing and laundering nanoparticle drug delivery vehicle formulations is a crucial step in developing alternative therapeutics. Nanoparticles, such as calcium phosphosilicate nanoparticles (CPSNPs), can effectively target specified disease tissue by bioconjugating with active target moieties. CPSNPs enhance the therapeutic window of active agents with maximum efficacy and minimal adverse events. This work focuses primarily on the application of CPSNPs for treatment of bone disease including cancer and severe bone defects requiring artificial implants. For translational research, large batch volumes and concentrations are required for efficacious delivery and cost-effective production. Van der Waals assisted microsphere laundering (VAML) of CPSNPs was analyzed as a means to replace traditional van der Waals high performance liquid chromatography (vdW-HPLC) laundering of CPSNPs. vdW-HPLC managed to produce 10’s of mL per day of processing; however, VAML has shown to produce liters of CPSNP suspension per day. CPSNP synthetic suspensions are loaded into a separatory funnel with polymeric microspheres for subsequent laundering and removal of cytotoxic precursors and spectator ions. The bond between microsphere and CPSNP is broken by collecting the CPSNP suspension in pH adjusted 70/30 EtOH/H2O (v/v) which effectively neutralizes the attractive force between the microspheres and CPSNPs. This work analyzed the number of washes with pH adjusted neat ethanol, the mass of microspheres used in relation to the volume of unlaundered CPSNPs loaded into the separatory funnel, and the volume concentration multiplier of the final eluted, laundered CPSNP suspension. Design of experiment (DoE) principles were applied to determine the appropriate parameters to maximize encapsulant concentration while minimizing residual surfactant concentration. VAML processed CPSNPs were found to have a minimal Igepal® CO-520 concentration and a maximum rhodamine WT (RhWT) concentration with 1 mg/mL microspheres, 5 washes, and a 25x volume concentration multiplier. Targeting metastatic breast cancer in bone was examined through in vitro experimentation. Prior to beginning the in vitro work, the bioconjugation of VAML processed CPSNPs was analyzed using a DoE factorial analysis. VAML creates CPSNPs with altered characteristics as a result of collecting smaller particles and increasing the particle number concentration in the final formulation. These changes resulted in the need to alter the bioconjugation process. A 1x CPSNP formulation was traditionally bioconjugated with 1 mg/mL 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and 10 mg/mL methoxy-poly(ethylene glycol) (mPEG). The concentration of CPSNPs, EDC, and mPEG were explored as a function of zeta potential neutralization as mPEG neutralizes the citrate surface functionalization (-30 to -40 mV) of synthesized CPSNPs. A surface charge of close to 0 mV was observed with a CPSNP concentration of 250x, an EDC concentration of 1 mg/mL, and a mPEG concentration of 100 mg/mL with a reaction that proceeded for 24 hours at 25 °C. The in vitro work incorporated CPSNPs bioconjugated with mPEG as a passive targeting control for αCD71, which is an antibody for the active targeting of overexpressed transferrin receptors on breast cancer. Using αCD71-CPSNPs with RhWT encapsulated, fluorescent micrographs were taken of a co-culture containing MDA-MB-231 human adenocarcinoma breast cancer on MC3T3-E1 osteoblasts. These images showed uptake of fluorescent CPSNPs into the breast cancer with no infiltration into the healthy bone cells. MTS assays were also analyzed to quantify cell death in single and co-culture. The cultures of both breast cancer and osteoblasts confirmed the microscopy results that targeted CPSNPs containing phosphorylated 5-FU (FdUMP) successfully knocked down the cancer cells and did no significant harm to the bone cells. However, the co-culture results showed ambiguous results via the MTS assay due to the chemotherapeutic killing breast cancer and the breast cancer harming bone cells. In addition to metastatic breast cancer in bone, CPSNPs were explored for their use in artificial bone constructs (ABCs) that would be ceramic based and serve as a clinical alternative to metallic implants. A 3D printing modality, direct ink writing (DIW), was applied to allow for patient specific implants to be printed with specific geometries to account for the intricate shape of bone defects. DoE principles were once again applied to ensure the commercial printer was capable of printing with the desired spatial resolution and accuracy required to mimic the microstructure of bone. With the intent of minimizing spatial resolution and printing with interconnected pores of 200 μm, the print parameters required for successful DIW of ABCs were a 30 wt% hydrogel paste, a 0.41 mm polypropylene nozzle, and a print speed of 175 mm/min. This work served as a proof of concept that an ABC doped with CPSNPs and hydroxyapatite could be 3D printed via DIW with the required geometric tolerances. Lastly, the 3D printing of ABCs was applied to a clinical setting. Working with doctors at Hershey Medical Center, a couple cases of bone defects where a ceramic composite construct would be beneficial compared to titanium were explored. In pediatric patients particularly, a biodegradable, incorporated component would allow for growth with the patient where titanium implants usually create additional surgical concerns as bones grow and fixation fails. Patient CT scans were edited in 3D modeling software to create a negative fill of the void in a humeral fracture. This fill served as a 3D model for printing a patient specific ABC that was later printed with fused deposition modeling along with the patient’s healthy bone. This work served to provide a full understanding of the life cycle of a biomedical implant including the regulatory and funding hurdles required for translational research.