*2.3. Generation and Characterization of Carbonate Apatite*

Carbonate apatite nanoparticles were manufactured via addition of Ca2<sup>+</sup> from 1 M CaCl2 stock solution to the bicarbonate-buffered cell culture medium (DMEM, pH adjusted to 7.5, containing 44 mM HCO3−), which already contained the third reactant (0.9 mM phosphate), followed by incubation at 37 ◦C for 30 min. As a result, microscopically visible carbonate apatite particles were formed through precipitation following nucleation in a supersaturated solution. Turbidity determination and size and zeta potential measurement of the variously formulated nanoparticles were employed for characterization of the resulting products [10].

#### *2.4. Complexation of Drugs and siRNAs with Carbonate Apatite*

Various concentrations of drugs and siRNAs (Table 1) were added in DMEM media (44 mM bicarbonate, pH 7.5) containing particular concentrations of CaCl2 and subjected to a 30-min incubation at 37 ◦C to allow formation of complexes.

### *2.5. In Vitro Viability Assay*

Cytotoxicity of carbonate apatite nanoparticles alone and also differently loaded NPs on human and murine breast cancer cell lines was assessed by MTT assay. Briefly, the cells from the exponential growth phase were seeded in 24-well plates (Griener, Frickenhausen, Germany) (approximately 50,000 cells/well) in DMEM with 10% FBS at 37 ◦C with 5% CO2. After 24 h, cells were exposed to various treatments for a consecutive period of 48 h. Two days later the viability was assessed by adding 50 μL MTT solution (5 mg/mL in phosphate buffered solution (PBS)) to each well and incubating for 4 h in dark. Then, the medium was removed and 300 μL DMSO was added to each well to dissolve the purple formazan crystals. Formazan quantification in the form of optical density (OD) was performed at test and reference wavelengths of 595 nm and 630 nm by a plate reader (benchmark plus, Bio Rad). Cell viability was determined using the following formula:

$$\text{Cell viability} \left( \% \right) \left( \text{CV} \right) = \frac{\text{OD}\_{\text{(treated)}} - \text{OD}\_{\text{(reference)}}}{\text{OD}\_{\text{(untreated)}} - \text{OD}\_{\text{(reference)}}} \times 100.$$

The reference was the optical density of DMSO only in the applied wavelengths.

Each experiment was done in triplicate and results are expressed as mean ± SD of % of cell viability. Subsequently, an increase in cytotoxic effect of the loaded NPs was calculated as follows:

Increase in toxicity (%) = CV baseline treatment − CV complete treatment,

where CV baseline treatment and CV complete treatment represent the cell viability resulting from the baseline treatment and complete treatment, respectively. In all bar charts (Supplementary Figures) displaying cell viability values, each two adjacent bars were compared together and an increase in toxicity was calculated for all different concentrations and expressed as mean ± SD.

#### *2.6. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (Sds-Page) and Western Blot*

Treated cells in each well of a 24-well plate were lysed by addition of 200 μL of whole cell IP-lysis buffer and protein sample was collected. Then, 5 μL solution was used for estimation of total protein content using the BSA assay kit according to instruction provided by the manufacturer (Quick-start Bradford protein assay kit, Bio Rad, Hercules, CA, USA).

Samples of the cell lysate containing equal amounts of total protein (e.g., 10 μg) were mixed with 10 μL of 10× loading dye and heated for 5 min at 95 ◦C and then resolved by SDS-PAGE using stain free mini protean SFX gels (10 wells) in 1X running buffer. In total, 7 μL of precision plus protein standards-dual color were used as a molecular weight marker to confirm the molecular weight of the proteins in the samples. The protein samples were transferred from gel to the 0.2-μm polyvinylidene difluoride (PVDF) membranes and attached using a trans-blot turbo transfer system (Bio Rad). Membranes were blocked in 5% skimmed milk in 1X TBST for an hour at room temperature.

The membrane was probed with indicated primary antibody (Table 2) overnight at 4 ◦C. Unbound primary antibodies were washed using 1X TBST buffer for 5 times, 5 min each, with gentle agitation. Blots were probed with horseradish peroxide conjugated secondary antibody (anti-rabbit IgG, 1:3000) for 1 h at room temperature. TBST was again used to remove excess secondary antibody by 5 wash cycles each 5 min long with gentle agitation. Clarity Western Enhanced Chemiluminescence (ECL) substrate (Bio-Rad) was applied onto the membrane in the dark for 5 min and the signals on the membrane were visualized via a Bio-Rad Gel documentation system.


**Table 2.** Information for the primary antibodies used for western blot in this study.

#### *2.7. Formulation of Particles for In Vivo Study*

The injectable nanoparticles were formulated in 100 μL of freshly prepared bicarbonated (44 mM) DMEM media to which CaCl2 was added. Samples were then incubated at 37 ◦C for 30 min followed

by maintenance on ice to prevent aggregation during injection. In drugcontaining samples, 1.25 mg/kg of Pac and 1 mg/kg of Doc were used prior to incubation. In case of using siRNAs, 50 nM of each siRNA was added to the media prior to incubation. The resulting therapeutics were used for iv treatment of animals.

#### *2.8. 4T1-Induced Breast Cancer Murine Model*

Female Balb/c mice with the age of 6 to 8 weeks and body weight of 15 to 20 g were used in this study.

Animals were maintained in a 12:12 light:dark condition and provided with food ab libitum and water. All experiments were performed in complete adherence to the regulations of Monash University Animal Welfare Committee. The details of the animal study were approved by Monash Animal Ethics Committee on 3 August 2012 with the project identification code of MARP/2012/087 under the title of "Delivery of anti-cancer drugs to breast cancer cells using nanoparticles". Tumor induction was performed via subcutaneous injection of 4T1 cells (in 100 μL PBS) on the mammary fat pad of mice. Injection day was considered as day 1 of the animal study. The development of tumor was regularly assessed through manual examination of the injection site. Randomization and treatment were carried out when the volume of the tumor reached an average of 13.20 <sup>±</sup> 2.51 mm<sup>3</sup> (Table 3). Treatment was administered via intravenous injection through the right or left caudal vein. Duration of study was 30 days, which involved close monitoring of the animals together with recording their body weights and tumor outgrowth every other day. The following formula was used for calculation of the tumor volume:

$$\text{Turnor volume} \left(\text{mm}^3\right) = \frac{\left(\text{Length} \times \text{Width}^2\right)}{2}$$


**Table 3.** Treatment groups used for in vivo study.

#### *2.9. Statistics*

For determining statistical significance of quantifications, student's *t*-test was used; all data are presented as mean ± SD. Data was considered significant for *p* values < 0.05.

#### **3. Results and Discussion**
