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. Author manuscript; available in PMC: 2022 Jun 1.
Published in final edited form as: Nanomedicine. 2021 Mar 13;34:102383. doi: 10.1016/j.nano.2021.102383

Preferential uptake of Antibody Targeted Calcium Phosphosilicate Nanoparticles By Metastatic Triple Negative Breast Cancer Cellsin Co-cultures of Human Metastatic Breast Cancer Cells plus Bone Osteoblasts

Karen M Bussard 1, Christopher M Gigliotti 2, Bernadette M Adair 3, Jenna M Snyder 4, Nicholas T Gigliotti 3, Welley S Loc 5, Zachary R Wilczynski 2, Zi-Kui Liu 3, Kacey Meisel 4, Cecilia Zemanek 4, Andrea M Mastro 4, Alison B Shupp 1, Christopher McGovern 6, Gail L Matters 6, James H Adair 2,3,7
PMCID: PMC8627646  NIHMSID: NIHMS1692764  PMID: 33722692

Abstract

Calcium phosphosilicate nanoparticles (CPSNPs) are bioresorbable nanoparticles that can be bioconjugated with targeting molecules and encapsulate active agents -and deliver them to tumor cells without causing damage to adjacent healthy tissue. Data obtained in this study demonstrated that an anti-CD71 antibody on CPSNPs, targets these nanoparticles to and enhances their internalization by triple negative breast cancer cells in-vitro. Caspase 3,7 activation, DNA damage, and fluorescent microscopy confirmed the apoptotic breast cancer response caused by targeted anti-CD71-CPSNPs encapsulated with gemcitabine monophosphate, the active metabolite of the chemotherapeutic gemcitabine used to treat cancers including breast and ovarian. Targeted anti-CD71-CPSNPs encapsulated with the fluorophore, Rhodamine WT, were preferentially internalized by breast cancer cells in co-cultures with osteoblasts. While osteoblasts partially internalized anti-CD71-GemMP-CPSNPs, their cell growth was not affected. These results suggest that CPSNPs may be used as imaging tools and selective drug delivery systems for breast cancer that has metastasized to bone.

Keywords: metastatic breast cancer, bone, osteoblast, gemcitabine, nanodelivery, nanoparticle

Graphical Abstract Text

Calcium phosphosilicate nanoparticles that directly target triple negative breast cancer cells using antibodies and deliver the chemotherapeutic gemcitabine. Bone is a preferred site of breast cancer metastases and once breast cancer invades the bone, overall survival and quality of living is poor. Currently there are no therapeutics available that selectively target the bone metastatic breast cancer cells while sparing endogenous bone stromal cells. We have developed an effective cargo encapsulation method utilizing calcium phosphosilicate nanoparticles encapsulated with the chemotherapeutic gemcitabine monophosphate, near-infrared imaging agent rhodamine WT, and bioconjugated with an antibody to CD71 to enhance selective targeting of breast cancer cells. A) αCD71-calcium phosphosilicate nanoparticles target metastatic breast cancer cells for subsequent uptake and dissolution of nanoparticles in late endosome and release of imaging agent and/or drug into the cytosol. B) Calcium phosphate nanocomposite particle showing co-encapsulation of rhodamine WT and gemcitabine monophosphate in a calcium phosphosilicate matrix, as well as surface bioconjugation of either 1) methoxy-polyethylene glycol with amine termination for passive targeting or 2) αCD71 for active targeting of transferrin receptors on breast cancer cells.

BACKGROUND

Despite the fact that the overall breast cancer (BrCa) survival rate has increased from 75% to 90% (1), there is still a need for enhanced clinical diagnosis and treatment. BrCa is the most common cancer for women in the United States and second most deadly cancer (1). Triple-negative breast cancer (TNBC), i.e. BrCa without estrogen and progesterone receptors and are HER2 protein negative, accounts for ~10–15% of diagnosed BrCa cases (2). When detected early, the 10-year recurrence-free rate of individuals with TNBC is ~97%.

TNBC has a higher incidence of recurrence and metastasis to secondary sites than hormone receptor positive BrCa (3). Bone is a preferential site of BrCa metastasis (4). Once BrCa metastasizes to bone the relative 5-year survival rate is <10% (1). Patients develop osteolytic lesions which hinder quality of life. Standard of care for bone metastatic TNBC is administration of chemotherapeutic agents, such as gemcitabine, taxanes, or anthracyclines, followed by treatment with platinum drugs when tumor response to standard chemotherapeutics fails (5).

Calcium phosphosilicate nanoparticles (CPSNP) are targetable, bioresorbable nanoparticles that have a long half-life within the bloodstream (6–11). CPSNPs can encapsulate active agents and be bioconjugated with biological molecules to deliver the particles to tumor cells without causing damage to healthy cells. We have shown that drug and bioimaging agent delivery via CPSNP encapsulation in-vitro and in-vivo (12): minimizes systemic and local side effects of the drug; permits controlled release of active agents directly into tumor cell cytosol via acidic pH changes in late stage endosomes and dissolution of the calcium phosphosilicate matrix (13, 14). The small diameters (10–25nm) for colloidally stable spherical nanoparticles promote long-term circulation of at least 96h providing greater opportunity for drug-CPSNPs to target diseased cells (7, 8, 10, 11). Furthermore, the dissolution products, Ca2+(aq), HxPO43-x (aq) and Si(OH)4 (aq), are benign and of negligible concentration relative to concentrations of chemical species in the cytosol of cells and human blood (1–5 mM: Ca2+ and HxPO43-x; ~0.5M: Si(OH)4) (15–19). CPSNPs that are not taken up by targeted tissue are benignly cleared intact as solid nanoparticles in the feces (10, 11, 20). Recently (6, 20, 21), we showed phosphate groups on the drug/imaging agent (e.g. GemMP) can be encapsulated, producing a large encapsulation efficiency (EE =(moles/final formulation)/(moles introduced during synthesis)) and permitting preparation of human dose-level formulations (i.e. 15 to 350 mg/m2). Animal trials for maximum tolerated dose via systemic IV injection do not have a MTD because the phosphorylated molecules rapidly clear with a short half-life (<15min). While current literature advises against use of phosphorylated drugs in humans due to the negative charge inhibiting trans-membrane transport and poor half-life (21–24), when drugs are encapsulated within CPSNPs, the short half-life of free phosphorylated drug is a feature that reduces side effects and is not a limitation.

The objective of this study was to develop a CPSNP encapsulated with a chemotherapeutic agent that preferentially targeted bone metastatic BrCa cells. Because all eukaryotic cells, especially rapidly dividing cancer cells, must maintain dNTP pools for DNA replication and repair, our focus was on incorporation of the phospho-gemcitabine analog gemcitabine monophosphate (GemMP). The prodrug, gemcitabine, is phosphorylated after passing across the cancer cell membrane. Thus, the phosphorylated form of Gem, GemMP disrupts replication by incorporating into DNA and inhibiting ribonucleotide reductase, which depletes cellular dNTP pools. This activity leads to stalled replication forks and induces DNA damage response (DDR) signaling pathway orchestrated by ATM/ATR kinases. Once activated, ATM phosphorylates downstream proteins, including histone H2A.X, which accumulates at sites of DNA damage, and promotes apoptosis through increased caspase signaling. Gemcitabine has been used to treat patients with metastatic BrCa, and clinical trials suggest it provides improved patient overall survival and progression-free survival without increased risk of adverse events (5, 25, 26).

While there are many examples of nanoparticle delivery systems being tested in the clinic to combat cancer (27); herein, our team developed an effective cargo encapsulation method utilizing CPSNPs encapsulated with the chemotherapeutic GemMP, near-infrared imaging agent rhodamine WT, and bioconjugated with an antibody to CD71 (Figure 1)(6, 20, 21). Transferrin receptor (TFR or CD71) is a cell-membrane-associated glycoprotein involved in cellular iron uptake and is overexpressed in breast, lung, ovarian, and brain cancer (28). Osteoblasts, however, downregulate the TFR as they differentiate and mineralize, making the TFR an ideal candidate as a target for therapeutics treating breast cancer metastases to bone (29, 30). We verified that CPSNPs bioconjugated with anti-CD71 and encapsulated with GemMP blocked BrCa cell growth and induce apoptosis through promotion of the DDR pathway and activation of executioner caspases. Furthermore, we showed anti-CD71-GemMP-CPSNPs did not negatively affect bystander stromal cells, specifically MC3T3E1 osteoblasts.

Figure 1. The mechanism of αCD71-CPSNPs nanoparticle uptake by BrCa cells.

Figure 1.

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A) αCD71-CPSNPs target metastatic breast cancer cells for subsequent uptake and dissolution of nanoparticles in late endosome and release of imaging agent and/or drug into the cytosol. B) Calcium phosphate nanocomposite particle cartoon showing co-encapsulation of RhWT (imaging agent) and GemMP (active pharmaceutical) in a calcium phosphosilicate matrix, as well as surface bioconjugation of either mPEG for passive targeting via the EPR effect or αCD71 for active targeting of transferrin receptors on breast cancer.

Materials and Methods

Detailed methods can be found in the online supplementary materials and methods.

Cells

Human MDA-MB-231 BrCa cells were a gift from Dr. Danny Welch (University of Kansas Cancer Center). MDA-MB-231-GFP cells, analogous to the parental MDA-MB-231 cell line, were engineered to express green fluorescent protein (GFP). Both cell types were maintained in DMEM without sodium pyruvate plus 5% (v/v) FBS, 1% penicillin 100 U/ml/streptomycin 100 μg/ml and 1% non-essential amino acids. MCF-7 human ER+ BrCa cells (31), were a gift from Dr. Mark Kester (University of Virginia). MCF-7 cells were maintained in EMEM (Gibco) supplemented with 10% FBS (Hyclone), 100 U/ml penicillin/100 mg/ml streptomycin, and 0.01μg/ml of recombinant human insulin (MP Biomedicals, Solon, OH). All BrCa cells were cultured in a humidified chamber with 5% CO2 at 37 °C.

Murine MC3T3-E1 osteoblasts, a gift from Dr. Norman Karin, Roswell Park Cancer Institute, were maintained in alpha-MEM plus 10% FBS and 1% penicillin/streptomycin, and cultured in a humidified chamber of 5% CO2 at 37 °C. MC3T3-E1 cells were seeded at 10,000 cells/cm2 in 96-well plates. 24h later, growth medium was replaced with a differentiation medium consisting of αMEM plus 10% (v/v) FBS, 1% penicillin/streptomycin, 10mM β-glycerophosphate, and 50μg/mL ascorbic acid. MC3T3-E1 cells were differentiated for 21 days, and medium replaced every 3rd day.

Cell Proliferation Assay

MC3T3-E1 cells were seeded at 10,000cells/cm2 in 96-well plates and differentiated for 21 days. Separately, MDA-MB-231 cells were seeded at 7,500cells/well in 96-well plates and allowed to adhere for 24h before exposure to CPSNP formulations. DPBS was added to 1% CPSNP formulations, then the mixture dried down under inert gas using aseptic techniques before resuspension in DPBS for in-vitro experiments (DPBS was added to prevent dissolution of CPSNPs when the 70:30 suspending medium was reduced to pure water). Cells were exposed to no treatment, vehicle (1x DPBS), free-GemMP, mPEG-Empty-CPSNP, anti-CD71-Empty-CPSNP, mPEG-GemMP-CPSNP, or anti-CD71-GemMP-CPSNP and assayed at 24, 48, 72, or 96h. MTS assay solution was added to treated wells at 20% (v/v), incubated for 90–180min, and absorbance measured at 490 nm with a Thermo Labsystems Multiskan Microplate Reader. The blank (medium and MTS assay reagents) was subtracted from absorbance values, and relative proliferation normalized to vehicle treatment (20% (v/v) DPBS).

Apoptosis and DNA Damage Response Assays

Caspase 3–7 Kit (#MCH100108) and Multi-Color DNA Damage Kit (#MCH 200107) were purchased from Luminex. For the DNA Damage Assay, 2×105 cells were plated in a 12-well plate and 4h after seeding, media were replaced with the indicated solution. 72h post treatment, cells were trypsinized and collected for analysis using the Muse cell analyzer (Millipore). For the Caspase 3–7 Assay, 1×105 cells were plated in a 24-well plate and 24h after seeding media were replaced with the indicated reagents. 96h post treatment, cells were trypsinized, collected and analyzed using the Muse system as directed. Data are presented as mean±standard error of the mean of at least three independent experiments. Comparison of the means between groups was carried out using unpaired two-tailed t-tests with Prism 6.0 software (GraphPad).

RESULTS

CPSNPS as Chemotherapeutic Nanoparticles to Target TNBC Cells

The growth of BrCa cell requires increased iron uptake mediated by overexpression of TFR1. Women with high TFR1 expression have poor prognosis (32). TFR1 levels can be upregulated by physiologic effectors including inflammation, oxidative stress, and hypoxia (33). These characteristics, as well as efficient cellular internalization upon ligand binding, make TFR an attractive receptor for targeted cancer nanotherapy in the context of metastatic disease. In this study, we developed nanoparticles that 1) targeted metastatic BrCa cells via a TFR antibody (anti-CD71) and 2) delivered physiologically-relevant amounts of chemotherapeutic drugs to cancer cells leaving stromal cells unharmed (Figure 1).

CPSNP characterization

CPSNPs were characterized using TEM photomicrographs for particle size distribution, and were evaluated using ImageJ (NIH) to determine area distribution for at least 300 particles (Figure 2A). The nanoparticles were calculated to have a lognormal mean (in real space) diameter of 25 nm, and lognormal standard deviation (sz) of 0.69. CPSNP maximum particle size was less than 60 nm. Based on the scale of the 85kDa a-CD71 in the range from 8–9nm, expected diameter would be ~33 nm for ~ 25 nm particles. However, with the low pressure vacuum required for TEM, surface proteins and mPEG dehydrate and reduce the scale observed by TEM. Therefore, the TEM in Figure 2 was taken of the citrate-CPSNPs that provides better contrast and reveals the size distribution of the small surface molecule, citrate (~0.5nm), on the calcium phosphosilicate nanoparticles with RhWT encapsulated.

Figure 2: Characterization of CPSNPs by TEM and Zeta Potential.

Figure 2:

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A) Representative TEM micrograph of a CPSNP formulation and lognormal particle size distribution of a Citrate-RhWT CPSNP suspension. The nanoparticles are well dispersed and have a lognormal mean (in real space) diameter of 25 nm and a lognormal standard deviation (sz) of 0.69. The log normal fit had an adjusted R2 value of 0.94 for n= 476. The maximum particle size is less than 60 nm. At 85kDa, anti-CD71 should have an oblate ellipsoid diameter of 8 to 9 nm diameter. In aqueous solutions, this gives a hydrodynamic diameter with the 2kDa PEG tether (at ~2nm) of over 30 nm (maximum diameter ~ 10+4+19 = ~32nm). However, both the PEG and proteins dehydrate in the low-pressure vacuum required for TEM analyses. The dehydrated organic matter also compromises resolution for the small-scale nanoparticles. B) Changes in zeta potential with each bioconjugation step verifies the change in surface chemistry for the nanomedical untargeted, methoxyPEG (mPEG) and targeted with aCD71. Additional details are provided in the Supporting Materials.

CPSNPs were also characterized using zeta potential as measured via ELS (Figure 2, Supplemental Figure 1). Zeta potential was analyzed to support successful bioconjugation of mPEG, cPEG, and anti-CD71 antibody to the surface of CPSNPs (Figure 2B, Supplemental Figure 1). Empty-CPSNPs showed when citrate is bioconjugated with mPEG, the zeta potential was neutralized from −57± 10mV to a value of −9 ± 11 mV, demonstrating successful conjugation as also evident by the shift in intensity distribution. cPEG-Empty-CPSNPs traditionally have not shown a shift in zeta potential as the carboxy group on both the citrate and cPEG dominates surface charge. The zeta potential for Empty-CPSNPs shifted from −57 ± 10 mV for citrate to −69 ± 5 mV for cPEG then −71± 4 mV for aCD71. The GEMMP-CPSNPs citrate to mPEG shifted from −46 ± 4 mV to 0 ± 1 mV and −37 ± 7 mV for the aCD71-GEMMP-CPSNP. Similarly, RhWT-CPSNPs had a zeta potential shift from −51 ± 10 mV for citrate to −69 ± 5 mV for cPEG, and then a surface charge for aCD71 of −67 ± 10 mV with a surface neutralization with mPEG of −13 ± 7 mV. The cPEG- generally did not have a significantly different zeta potential than the anti-CD71 antibody conjugated CPSNPs. Increasing the cPEG to anti-CD71 antibody ratio during the bioconjugation may account for the lack of a significant shift in zeta potential.

GemMP decreased BrCa cell proliferation in-vitro in a dose-dependent manner

Dose-response toxicity of GemMP on MDA-MB-231 cells was evaluatedin-vitro (Figure 3). At 72h post-GemMP exposure, a dose-dependent decrease in MDA-MB-231 cell proliferation was observed (Figure 3A). Compared with exposure to vehicle, statistically significant decreases in BrCa cell proliferation were found with as little as 10nM GemMP. The largest change in BrCa cell proliferation, ~10-fold decrease, was observed with addition of 10mM GemMP. For MDA-MB-231 cells, the LD50 of GemMP was determined to be 100nM.

Figure 3. Breast cancer cell growth was decreased after exposure to free GemMP or to GemMP-CPSNPs in-vitrowhile osteoblast cells are unaffected.

Figure 3.

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All cells were treated as indicated for 72 h; cell growth was expressed as percent of vehicle (VEH), 20% (v/v) DPBS. A) Relative proliferation of MDA-MB-231 cells after treatment with free GemMP. Data is represented as mean ± 95% confidence interval. ** p ≤ 0.01, *** p ≤ 0.001 compared to vehicle. MDA-MD-231 cell growth was reduced in a dose dependent manner by GemMP concentrations between 10 nM and 10 μM, and the LD50 for GemMP was 100 nM. B) Compared to VEH treated cells or cells treated with anti-CD71 targeted empty-CPSNPs, MDA-MB-231 cells treated with 100 nM of free GemMP, or with 100 nM, 1mM, or 10 mM anti-CD71-GemMP-CPSNPs demonstrated a dose-dependent reduction in cell growth. Lower concentrations of anti-CD71-GemMP-CPSNPs, including 1nM and 10 nM, were less effective. **p<0.01 compared with vehicle and error bars indicate the 95% CI. Hatch marks indicate GemMP treatments; while dark bars indicate treatment with CPSNPs. C) Growth of MCF-7 human breast cancer cells was blocked by both free GemMP (100 nM) and anti-CD71-GemMP-CPSNPs (100 nM), ***p<0.001 compared to vehicle. In this in-vitroassay, untargeted GemMP-CPSNPs were as effective in blocking growth as anti-CD71-targeted GemMP CPSNP treatments. D) Unlike breast cancer cells, growth of MC3T3-E1 human osteoblasts treated with GemMP or with GemMP loaded CPSNPs, either with or without anti-CD71 targeting, was not significantly different from vehicle controls.

Anti-CD71-GemMP-CPSNPs effectively blocked BrCa cell proliferation in-vitro

Previously our group demonstrated CPSNPs can encapsulate phosphorylated, bioactive analogs of chemotherapeutic drugs including FdUMP and GemMP (20). To demonstrate that anti-CD71-CPSNPs effectively deliver GemMP to BrCa cells in-vitro and reduce cell viability, MDA-MB-231 cells were treated with various concentrations of anti-CD71-GemMP-CPSNPs, free-GemMP, and vehicle and empty-CPSNP controls, for 72h (Figure 3B). Compared to vehicle, anti-CD71-Empty-CPSNPs had no effect on cell viability verifying that CPSNPs themselves were non-toxic to BrCa cells. Although 1nM and 10nM anti-CD71-GemMP-CPSNPs did not significantly reduce MDA-MB-231 proliferation, 100nM anti-CD71-GemMP-CPSNPs were as effective as 100nM free-GemMP in blocking BrCa cell growth. Higher doses of anti-CD71-GemMP-CPSNPs, up to 1μM and 10μM, were even more efficacious against MDA-MB-231 cells (Figure 3B).

The experiment was repeated using MCF-7 ER+ cells exposed for 72h to anti-CD71- and mPEG-GemMP-CPSNPs (100nM), 100nM free-GemMP, and vehicle and empty-CPSNPs (Figure 3C). Similar to MDA-MB-231 cells, empty-CPSNPs had no effect on MCF-7 cell growth. Statistically significant reductions were observed in MCF-7 cell proliferation upon exposure to 100nM free-GemMP. Interestingly, 100nM anti-CD71-GemMP-CPSNPs were as effective at reducing ER+ cell proliferation as 100nM mPEG-GemMP-CPSNPs. These data suggest untargeted- and anti-CD71-targeted-CPSNPs effectively delivered GemMP to BrCa cells in-vitro, with ER+ and ER- cells responding similarly.

MC3T3-E1 osteoblasts were treated under the same conditions as MCF-7 cells (Figure 3D). Compared to vehicle, no statistically significant change in MC3T3-E1 osteoblast proliferation was observed after exposure to any CPSNPs, indicating that the CPSNPs, even with the chemotherapeutic present, are non-toxic to MC3T3-E1 osteoblasts.

BrCa apoptosis was increased upon exposure to free-GemMP or GemMP-CPSNPs in-vitro

To determine if the decrease in BrCa cell proliferation upon exposure to anti-CD71-GemMP-CPSNPs was due to an increase in BrCa cells undergoing cell death, MDA-MB-231 cells were exposed for either 48, 72, 96, or 144h to 100nM targeted-GemMP-CPSNPs, anti-CD71-empty-CPSNP, mPEG-GemMP-CPSNPs, mPEG-empty-CPSNPs, free-GemMP, vehicle control (20% DPBS), or no treatment, and assayed for BrCa cell apoptosis. 144h post-exposure, a statistically significant increase in BrCa cell apoptosis was observed when MDA-MB-231-GFP cells were exposed to free-GemMP, mPEG-GemMP-CPSNPs, or anti-CD71-GemMP-CPSNP (Figure 4A). In this in-vitro assay, mPEG-GemMP CPSNPs were as effective in increasing BrCa cell apoptosis as anti-CD71-GemMP CPSNPs. Importantly, fluorescent microscopy revealed that MDA-MB-231-GFP cells exposed to free-GemMP, mPEG-GemMP-CPSNPs, or anti-CD71-GemMP-CPSNP exhibited similar morphology whereby exposure to GemMP caused BrCa cell swelling followed by apoptosis (Figure 4B-H). MDA-MB-231-GFP cells given no treatment, or exposed to vehicle (DPBS), anti-CD71-Empty-CPSNPs, or mPEG-Empty-CPSNPs did not exhibit cellular swelling and showed normal morphology (Figure 4B-H). The same experiment was carried out at 48, 72, and 96h; however, no statistically significant difference was observed (data not shown). These data suggest BrCa cell apoptosis increased upon exposure to GemMP-encapsulated-CPSNPs in-vitro.

Figure 4. Breast cancer cell apoptosis is increased after exposure to free GemMP or to GemMP CPSNPs in-vitro.

Figure 4.

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MDA-MB-231-GFP breast cancer cells were treated as indicated for 144 h and cell apoptosis is expressed as percent of vehicle, 20% (v/v) DPBS. A) Compared to vehicle (VEH) treated cells, cells treated with mPEG untargeted empty CPSNPs, or cells treated with anti-CD71 targeted empty CPSNPs, MDA-MB-231 human breast cancer cells treated with 100 nM of free GemMP, 100 nM of mPEG GemMP-CPSNPs, or 100 nM anti-CD71 GemMP CPSNPs demonstrated an increase in cellular apoptosis. ***p=0.0001, ****p<0.0001 compared with vehicle and error bars indicate STDEV. Hatch marks or cross marks indicate GemMP treatments, while dark bars indicate treatment with CPSNPs. B-H) MDA-MB-231-GFP breast cancer cells were exposed to B) no treatment (NT), C) vehicle (20% DPBS), D) 100 nM free GemMP, E) 100 nM mPEG Empty CPSNPs, F) 100 nM mPEG GemMP CPSNPs, G) 100 nM anti-CD71 Empty CPSNPs, or H) 100 nM anti-CD71 GemMP CPSNPs for 144 hours then imaged via fluorescent microscopy (FITC). Apoptosis of MDA-MB-231 human breast cancer cells is increased by free GemMP (100 nM), mPEG GemMP CPSNPs (100 nM), and anti-CD71 GemMP CPSNPs (100 nM). In this in-vitroassay, mPEG (untargeted) GemMP CPSNPs were as effective in increasing breast cancer cell apoptosis as anti-CD71 targeted GemMP CPSNP treatments. Three biological replicates were carried out per experimental condition; shown are representative images. Scale bar = 100 μm.

DNA Damage Repair pathways were activated by GemMP-CPSNPs

To further explore whether DNA damage induced by GemMP-CPSNPs was responsible for caspase activation and apoptosis, markers of DDR pathway activation were assessed in MDA-MB-231 cells treated with free-GemMP or with CD71-targeted-GemMP-CPSNPs. Similar to caspase activation assays, the percentage of cells with DNA damage, measured by an increase in phosphorylated-ATM and phosphorylated-histone H2A.X, was not significantly increased in vehicle or empty-CPSNP-treated controls compared to untreated cells (Figure 5A). After 72h of treatment, both free-GemMP and anti-CD71-GemMP-CPSNPs were equally effective at inducing DDR pathway activation. The significant increase in phospho-ATM and phospho-H2A.X in both free-GemMP and anti-CD71-targeted-GemMP-CPSNP treated cells is consistent with the known mechanism of action of gemcitabine: stimulating double strand DNA breaks caused by stalled replication forks and incorporation of the modified nucleotide into DNA strands.

Figure 5: Targeted-GemMP-CPSNPs effectively activated markers of DNA damage in in-vitro treated MDA-MB-231 cells.

Figure 5:

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A) DNA damage response activation is a measure that combines the amount of ATM phosphorylation and H2A.X histone phosphorylation. After 72 h of incubation, untreated cells (NT), vehicle treated cells (VEH), and cells treated with CPSNPs without drug (Empty) showed no increase in DNA damage. However, compared with those negative controls, both free GemMP (100 nM) and targeted-GemMP loaded CPSNPs (100 nM) were equally effective in increasing the percentage of cells that were positive for DNA damage (***p<0.001, error bars indicate the SEM). Hatch marks indicate GemMP treatments, while dark bars indicate treatment with CPSNPs. B) Targeted-GemMP-CPSNPs triggered executioner caspase activation as effectively as free GemMP. At 96 h after treatment, both 100nM and 10 nM concentrations of free GemMP and targeted-GemMP-CPSNPs increased the percentage of caspase 3/7 positive MDA-MB-231 cells in a dose dependent manner. In comparison, 1 nM concentrations of either free GemMP or targeted-GemMP-CPSNPs were no more efficacious in activating caspases than were the negative controls (NT=untreated, VEH=vehicle and Empty=empty-CPSNPs). **p<0.01 compared to untreated cells and error bars indicate the SEM. Hatch marks indicate GemMP treatments, while dark bars indicate treatment with CPSNPs.

Targeted GemMP-CPSNPs activated pro-apoptotic caspases in-vitro

To confirm that reduced cell viability in GemMP-CPSNP treated MDA-MB-231 cells was due to increased apoptosis, activation of caspases 3,7 in treated and untreated cells was determined. While vehicle and empty-CPSNP treated cells had no increase in cleaved caspase positive cells compared to untreated controls, cells treated with free-GemMP or anti-CD71-GemMP-CPSNPs showed a dose-dependent increase in caspase 3/7 activation (Figure 5B). 100nM and 10nM doses of free-GemMP and anti-CD71-GemMP-CPSNPs significantly increased percentage of caspase 3/7 positive cells. However, at the 1nM dose, neither free-GemMP nor anti-CD71-GemMP-CPSNPs increased caspase activation compared to negative controls. No statistical difference between equivalent doses of free-GemMP and anti-CD71-GemMP-CPSNPs was observed. Therefore, anti-CD71-targeted-GemMP-CPSNPs were as effective as free-GemMP in activating executioner caspases associated with the intrinsic apoptotic pathway.

Human BrCa cells preferentially internalized targeted-CPSNPs

To confirm human BrCa cells internalized CPSNPs, MDA-MB-231 cells were exposed to anti-CD71-RhWT-CPSNPs or mPEG-RhWT-CPSNPs for 24h, then cells imaged by fluorescent microscopy. MDA-MB-231 cells preferentially internalized anti-CD71-RhWT-CPSNPs (Supplemental Figure 2A-B) as opposed to little to no uptake of mPEG-RhWT-CPSNPs (Supplemental Figure 2C-D). These results suggested that anti-CD71-antibody targeting of CPSNPs enhanced internalization by BrCa cells.

Osteoblast cells minimally internalized targeted CPSNPs

To determine whether osteoblasts internalize CPSNPs, 21-day differentiated MC3T3-E1 osteoblasts were incubated for 24h with anti-CD71-RhWT-CPSNPs or mPEG-RhWT-CPSNPs, then imaged via fluorescent microscopy. Interestingly, MC3T3-E1 osteoblasts minimally internalized anti-CD71-RhWT-CPSNPs (Supplemental Figure 3A-C) versus little to no uptake of mPEG-RhWT-CPSNPs (Supplemental Figure 3D-F). These results suggested that MC3T3-E1 osteoblasts minimally internalized anti-CD71-RhWT-CPSNPs, but not mPEG-RhWT-CPSNPs in-vitro.

Anti-CD71-RhWT-CPSNPs were preferentially taken up MDA-MB-231 cells in co-culture with MC3T3-E1 osteoblasts

To determine the effect of anti-CD71-RhWT-CPSNPs on BrCa cells and osteoblasts in co-culture, MDA-MB-231-GFP cells were added to cultures of 21-day differentiated MC3T3-E1 osteoblasts. After 24h, anti-CD71-RhWT-CPSNPs were added to co-cultures, then cells imaged 24h later. MDA-MB-231-GFP cells preferentially internalized anti-CD71-RhWT-CPSNPs, while little to no uptake of anti-CD71-RhWT-CPSNPs by MC3T3-E1 osteoblasts was observed (Figure 6A-D). These results indicated that anti-CD71-targeting-CPSNPs enhanced internalization by BrCa cells while MC3T3-E1 osteoblasts did not internalize anti-CD71-targeting-CPSNPs.

Figure 6: BrCa cells preferentially internalized targeted anti-CD71-RhWT-CPSNPs in co-cultures of MDA-MB-231 cells and MC3T3-E1 osteoblasts.

Figure 6:

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MDA-MB-231 breast cancer cells were exposed to 1 μM of A-D) anti-CD71-RhWT-CPSNPs for 24 hours then imaged via phase and fluorescent microscopy at 20x (A-B) and 40x magnification (C-D). A, C) merged phase and fluorescent images; B, D) phase images. MDA-MB-231 breast cancer cells preferentially took up anti-CD71-RhWT-CPSNPs (A-D) in co-cultures with MC3T3-E1 osteoblasts in-vitro. MC3T3-E1 osteoblast targeted particle uptake was minimal.

DISCUSSION

This work demonstrated that a CD71 antibody, used as an active targeting molecule on CPSNP surface, delivered cargo to metastatic BrCa cells co-cultured with bone cells. Importantly, metastatic BrCa cells preferentially internalized anti-CD71-CPSNPs compared to osteoblasts which showed low uptake (Figure 6, Supplemental Figures 2-3). Data indicated that, upon internalization with GemMP-CPSNPs, both TNBC and ER+ metastatic BrCa cell growth decreased in a dose-dependent manner (Figure 3B-C), and cells underwent apoptosis leading to increased DNA damage and activity of caspases 3,7 (Figures 4–5). Of clinical significance, differentiated osteoblasts, which closely represent bone in a physiological setting, cultured alone or with BrCa cells, were unaffected by targeted or untargeted GemMP-CPSNPs (Figure 3D, Supplemental Figure 3) suggesting that the concentration of GemMP needed to achieve a therapeutic effect on BrCa cells (100nM) did not harm bone cells. Importantly, our study specifically examined osteoblasts in their mature, differentiated state, whereby they lay down bone matrix, and is most representative of a physiological setting. In this state, osteoblast proliferation and DNA synthesis is decreased, and instead the osteoblast undergoes a differentiation process whereby they produce proteins important for mature bone matrix, including bone sialoprotein and osteocalcin (34, 35). Of note, GemMP specifically acts on cells that are actively proliferating and synthesizing DNA, that is, cells in the S phase of cell cycle (24). Since differentiated osteoblasts are in a state of growth arrest with inhibited DNA synthesis, any effects of targeted GemMP-CPSNPs would be negated. Thus, it may be possible to treat BrCa that has metastasized to bone while minimizing unwanted damage to surrounding tissue.

Nanoparticles have become increasingly popular as drug delivery systems in medicine, being utilized in cardiovascular disease, AIDS, and cancer therapy among others (36–38). An ideally designed nanoparticle will be capable of targeted cell tropism, effective cargo delivery, while minimizing damage to nearby healthy tissue (38). Herein we designed anti-CD71-CPSNPs as targeting agents (Figure 1). As controls, we tested the bioreactivity of empty-CPSNPs with MDA-MB-231 ER- BrCa cells, MCF-7 ER+ BrCa cells, and MC3T3-E1 osteoblasts. We found empty-CPSNPs had no effect on ER+ or ER- BrCa cell or osteoblast cell viability (Figure 3). Furthermore, there was no difference in the amount of DNA damage or percent activity of caspases 3,7 between empty-CPSNPs and no treatment or vehicle control (20% DPBS) (Figure 5). These data suggested that CPSNPs caused no pro-apoptotic or DDR by themselves.

We also effectively loaded CPSNPs with GemMP (Figure 1)(5). We found GemMP-loaded-CPSNPs were just as effective at reducing ER+ or ER- BrCa cell growth, increasing apoptosis and DNA damage, and increasing the amount of caspase 3,7 positive cells as free (unencapsulated) GemMP between 72–144h after exposure to CPSNPs or free-GemMP (Figure 3–5). In fact, no statistically significant differences were observed in percent of BrCa cells with DNA damage 72h after exposure to 100nM free-GemMP or 100nM anti-CD71-GemMP-CPSNPs (Figure 5A). Similarly, no statistically significant differences were observed in the percent of caspase 3,7 positive BrCa cells 96h after exposure to 10nM or 1nM free-GemMP or anti-CD71-GemMP-CPSNPs (Figure 5B). These results suggest the mechanism of encapsulated-GemMP was identical to free-GemMP.

Chemotherapeutics are usually considered the first treatment choice for individuals who have progressed to metastatic disease, have become resistant to hormone therapy, or present with hormone receptor negative metastatic BrCa (39, 40). Systemic chemotherapy may also be utilized as an adjuvant treatment in metastatic BrCa patients who were at high risk of recurrence. However, systemic chemotherapy has deleterious side effects, including nausea, vomiting and poor quality of life, as well as causing harm to normal cells (39, 40). We developed CPSNPs encapsulated with GemMP, an anti-metabolite, deoxycytidine-analogue, and nucleotide analogue that inhibits DNA synthesis (5, 41, 42). GemMP has limited toxicity and high efficacy, making it an ideal chemotherapeutic agent (42). Of clinical significance, both hormone receptor negative (MDA-MB-231) and hormone receptor positive (MCF-7) BrCa cells exhibited decreased proliferation upon internalization of either untargeted-GemMP- or anti-CD71-targeted-GemMP-CPSNPs (Figure 3). TNBC, that is hormone receptor and HER2 negative BrCa, is an extremely aggressive form of BrCa due to its ability to rapidly proliferate, metastasize to many organs including lung, liver, and bone, and high resistance to hormone receptor treatments (43, 44). On the other hand, BrCa that is hormone receptor positive, including ER+ BrCa, tends to grow more slowly over time and is responsive to hormone therapy (45). The 5-year relative survival rate for ER+ BrCa is high (>90%), where the majority of patients live on average for 19–21 years post-diagnosis (45, 46). This is compared to a 5-year relative survival rate of 60% for TNBC (43). However, over time, ER+ BrCa may also recur in secondary sites including the skeleton (45). Thus, drug-loaded-CPSNPs may be used to treat both ER+ and triple negative cancers. Furthermore, and of particular note, we found free-GemMP exhibited low toxicity to MC3T3-E1 osteoblasts in culture (Figure 3D) suggesting that unwanted damage to normal tissue may be minimized. While we chose to encapsulate GemMP into CPSNPs for the current study, it would be possible to encapsulate other drugs, including platinum drugs (47, 48) or gene therapies (5), alone or in combination, to selectively target metastatic BrCa cells.

Here, we utilized a monoclonal antibody against the TFR, anti-CD71, as an active targeting molecule on the CPSNP surface to enhance nanoparticle up-take by BrCa cells. Importantly, the TFR is overexpressed in BrCa cells, but downregulated in differentiated osteoblasts, making it an excellent choice as a targeting molecule on our CPSNP surface to directly attack BrCa cells but spare endogenous osteoblasts (28–30). In addition to targeted nanoparticle tropism as a means of chemotherapeutic cargo delivery, TFR-targeted CPSNPs may also represent a new tool for molecular imaging of metastatic BrCa in-vivo, which could better identify metastatic lesions or gauge disease response (49). Here, we utilized RhWT as an imaging agent to confirm cellular internalization of RhWT-CPSNP nanoparticles in-vitro by metastatic BrCa cells alone or in co-culture with osteoblasts (Figure 6, Supplemental Figures 2-3). However, other imaging agents may be utilized in the future. Others have utilized 89Zr-transferrin PET, which noninvasively images TFR, to successfully detect tumors in pre-clinical TNBC models using MDA-MB-231 cells (50).

Overall, our results indicate anti-CD71-GemMP-CPSNPs facilitate MDA-MB-231 cell death by means of gemcitabine-induced DNA replication stress and caspase-mediated apoptosis.

Supplementary Material

1. Supplemental Figure 1. Mean Zeta potential of CPSNPs as a function of surface functionalization.

In-vitroBrCa treatments: A) Empty-CPSNP, control; C) GemMP-CPSNP, drug-loaded; E) RhWT-CPSNP, visualize cellular uptake. Error bars, 95% CI for 5 data points collected using ELS on Brookhaven Omni. Surface neutralization was seen with mPEG conjugation. A minor, yet significant, shift in zeta potential was evident with cPEGylation. No significant difference was seen with cPEG-αCD71 conjugation (B, D, F). Relative intensity as a function of zeta potential for surface functionalizations. Breadth of distribution depicts effectiveness of bioconjugation. * p ≤ 0.05.

2. Supplemental Figure 2: MDA-MB-231 cells take up CPSNPs.

MDA-MB-231 breast cancer cells were exposed to 1 μM of A-B) anti-CD71-RhWT-CPSNPs or C-D) mPEGRhWT-CPSNPs for 24 hours then imaged via phase and fluorescent microscopy. A and C) Fluorescent microscope images for TRITC; B and D) merged phase and fluorescent images. MDA-MB-231 breast cancer cells preferentially take up anti-CD71RhWT-CPSNPs (A, B), but not mPEG-RhWT-CPSNPs (C, D).

3. Supplemental Figure 3: MC3T3-E1 osteoblasts partially internalized CPSNPs.

MC3T3-E1 osteoblasts were exposed to 1 μM of A-C) anti-CD71-RhWT-CPSNPs or D-F) mPEG-RhWT-CPSNPs for 24 hours then imaged via phase and fluorescent microscopy. A) Phase microscope image; B and E) fluorescent microscope images for TRITC; D)fluorescent microscope images for FITC; C and F) merged phase and fluorescent images. MC3T3-E1 osteoblasts partially internalized anti-CD71-RhWT-CPSNPs (A-C), but not mPEG-RhWT-CPSNPs (D-F) in-vitro.

4

ACKNOWLEDGMENTS

This study was funded in part byNational Institutes of Health (NIH) grants R01CA167535 (GLM and JHA) and R21CA170121 (GLM) from the National Cancer Institute (NCI), NIH, NCI CA178177–05 (KMB); Pennsylvania Department of Health, Tobacco CURE funds SAP#4100072562 (GLM and JHA), and SAP#4100072566 (KMB); and The Pennsylvania Breast Cancer Coalition (KMB). CMG and WSL were supported, in part, by grants UL1 TR000127 and TL1 TR000125 from the National Center for Advancing Translational Sciences (NCATS). The department of Materials Science and Engineering at Penn State disclaims responsibility for any analyses, interpretations or conclusions. Penn State Research Foundation has licensed CPSNP technology to Keystone Nano, Inc. (PA, USA). JHA is a co-founder of PandreaBio (formerly Keystone Nano) and CSO. All other authors declare no conflicts of interest.

ABBREVIATIONS

5-FdUMP

5-Fluoro-2’-deoxyuridylate

Alpha-MEM

Alpha Modification of Minimal Essential Medium

anti-CD71

Human antigen for CD71 receptor in MDA-MB-231 cell membrane

ATM

Ataxia-telangiectasia mutated kinase related to the DNA damage response

ATR

ATM- and Rad3 related to the DNA damage response

BrCa

breast cancer

cPEG

Carboxy-Polyethylene Glycol with amine termination

CPSNP

Calcium Phosphosilicate Nanoparticle

DDR

Deoxyribonucleic Acid Damage Response

DLS

Dynamic Light Scattering

DMEM

Dulbecco’s Minimal Essential Medium

DNA

Deoxyribonucleic Acid

DDR

DNA damage response

dNTP

Deoxynucleoside triphosphate

DPBS

Dulbecco’s Phosphate Buffered Saline

ELS

Electrophoretic Light Scattering

ER+

Estrogen Receptor Positive

FBS

Fetal Bovine Serum

Gem

Gemcitabine

GemMP

Gemcitabine Monophosphate

GFP

Green Fluorescent Protein

Her2

Human Epidermal Growth Factor Receptor 2

MCF-7

Human Estrogen Receptor Positive Breast Cancer Cells

MDA-MB-231

Human Triple Negative Breast Cancer Cells

mPEG

Methoxy-Polyethylene Glycol with amine termination

NT

No Treatment

SEM

Standard Error of the Mean

TEM

Transmission Electron Microscopy

TFR

Transferrin Receptor

TNBC

Triple Negative Breast Cancer

Footnotes

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1. Supplemental Figure 1. Mean Zeta potential of CPSNPs as a function of surface functionalization.

In-vitroBrCa treatments: A) Empty-CPSNP, control; C) GemMP-CPSNP, drug-loaded; E) RhWT-CPSNP, visualize cellular uptake. Error bars, 95% CI for 5 data points collected using ELS on Brookhaven Omni. Surface neutralization was seen with mPEG conjugation. A minor, yet significant, shift in zeta potential was evident with cPEGylation. No significant difference was seen with cPEG-αCD71 conjugation (B, D, F). Relative intensity as a function of zeta potential for surface functionalizations. Breadth of distribution depicts effectiveness of bioconjugation. * p ≤ 0.05.

NIHMS1692764-supplement-1.tif (26.9MB, tif)
2. Supplemental Figure 2: MDA-MB-231 cells take up CPSNPs.

MDA-MB-231 breast cancer cells were exposed to 1 μM of A-B) anti-CD71-RhWT-CPSNPs or C-D) mPEGRhWT-CPSNPs for 24 hours then imaged via phase and fluorescent microscopy. A and C) Fluorescent microscope images for TRITC; B and D) merged phase and fluorescent images. MDA-MB-231 breast cancer cells preferentially take up anti-CD71RhWT-CPSNPs (A, B), but not mPEG-RhWT-CPSNPs (C, D).

NIHMS1692764-supplement-2.tif (16MB, tif)
3. Supplemental Figure 3: MC3T3-E1 osteoblasts partially internalized CPSNPs.

MC3T3-E1 osteoblasts were exposed to 1 μM of A-C) anti-CD71-RhWT-CPSNPs or D-F) mPEG-RhWT-CPSNPs for 24 hours then imaged via phase and fluorescent microscopy. A) Phase microscope image; B and E) fluorescent microscope images for TRITC; D)fluorescent microscope images for FITC; C and F) merged phase and fluorescent images. MC3T3-E1 osteoblasts partially internalized anti-CD71-RhWT-CPSNPs (A-C), but not mPEG-RhWT-CPSNPs (D-F) in-vitro.

NIHMS1692764-supplement-3.tif (26.8MB, tif)
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NIHMS1692764-supplement-4.docx (29.7KB, docx)

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