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Precision Nanomedicine

The official journal of CLINAM

Precision Nanomedicine (PRNANO) is a fully open access, peer-reviewed international society journal which promotes all practical, rational, and progressive aspects of theory and practice of nanomedicine, from basic research through translational and clinical aspects including commercialization. PRNANO provides a forum with reliable content and quick turnaround time. Papers are published continuously and are organized into quarterly issues.

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Nanoparticle-Encapsulated Doxorubicin Demonstrates Superior Tumor Cell Kill in Triple Negative Breast Cancer Subtypes Intrinsically Resistant to Doxorubicin
- Krausz AE, Adler BL, Makdisi J, Schairer D, Rosen J, Landriscina A, Navati M, Alfieri A, Friedman JM, Nosanchuk JD, Rodriguez-Gabin A, Ye KQ, McDaid HM, Friedman AJ.
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Nanoparticle-Encapsulated Doxorubicin Demonstrates Superior Tumor Cell Kill in Triple Negative Breast Cancer Subtypes Intrinsically Resistant to Doxorubicin

Research Article

Aimee E. Krausz1#, Brandon L. Adler1#, Joy Makdisi1, David Schairer1, Jamie Rosen1, Angelo Landriscina1, Mahantesh Navati2, Alan Alfieri2, Joel M. Friedman2, Joshua D. Nosanchuk3, Alicia Rodriguez-Gabin4, Kenny Q Ye5, Hayley M. McDaid4,6*, and Adam J. Friedman1,2,7 * [1]

1Division of Dermatology, Department of Medicine, 2Department of Physiology and Biophysics, 3Department of Microbiology & Immunology, 4Department of Molecular Pharmacology, 5Department of Epidemiology and Population Health, 6Department of Medicine (Oncology), Albert Einstein College of Medicine, Bronx, New York, USA, 7Department of Dermatology, George Washington School of Medicine, Washington, DC, USA

#These authors contributed equally to this work

*Co-senior authorship

Submitted: July 18, 2018; Accepted: October 22, 2018; Posted October 27, 2018

Abstract

The effect of size and release kinetics of doxorubicin-nanoparticles on anti-tumor efficacy was evaluated in a panel of human cancer cell lines, including triple-negative breast cancer (TNBC) cells that frequently demonstrate resistance to doxorubicin. Different nano-formulations of sol-gel-based Doxorubicin containing nanoparticles were synthesized. Increased cell kill in chemorefractory triple-negative breast cancer cells was associated with the smallest size of nanoparticles and the slowest release of Dox. Modeling of dose-response parameters in Dox-sensitive versus Dox-resistant lines demonstrated increased EMax and area under the curve in Dox-resistant mesenchymal TNBC cells, implying potentially favorable activity in this molecular subtype of breast cancer. Mesenchymal TNBC cells demonstrated a high rate of fluorescent bead uptake suggestive of increased endocytosis, which may partially account for the enhanced efficacy of Dox-np in this subtype. Thus, manipulation of size and release kinetics of this nanoparticle platform is associated with enhanced dose-response metrics and tumor cell kill in therapeutically recalcitrant TNBC cell models. This platform is easily customizable and warrants further exploration.

Keywords:

  • Experimental therapeutics
  • Nanoparticles
  • Triple-negative breast cancer
  • Doxorubicin
  • Drug resistance
  • Dose-response relationship

Abbreviations:

  • pCR: Pathologic Complete Response
  • TNBC: Triple-Negative Breast Cancer
  • AUC: Area under the curve
  • Dox: Doxorubicin
  • Dox-np: Doxorubicin-Nanoparticles

Purpose and Rationale

The purpose of this study was to synthesize and characterize a nanoparticle carrier for doxorubicin (Dox) and evaluate its biologic activity against a range of human cancer cell lines, focusing on TNBC in particular. Our goal was to investigate the pre-clinical anti-tumor activity of Dox-np relative to the parent drug.

Introduction

Doxorubicin, is a well-known anthracycline used primarily in combination chemotherapy for numerous malignancies, notably breast cancer, particularly triple-negative breast cancer (TNBC). The majority of TNBC patients receive a combination of taxane, doxorubicin and cyclophosphamide (TAC), as preoperative neoadjuvant chemotherapy, of which 25–45% achieve pathologic complete response (pCR) and have excellent long-term prognosis (1). Patients who fail TAC have poor prognosis with limited post-operative treatment options available (2). Response to TAC is predominately influenced by the molecular subtype of TNBC (3-5) of which mesenchymal (M) and BL2 tumors have the poorest response and long-term survival due to metastatic biology (2, 4), highlighting an unmet clinical need.

Encapsulation of Dox in a biocompatible nanoparticle platform could expand its narrow therapeutic index (6), enabling slow and sustained release of contents. This has the potential to limit toxicity since the theoretical maximum amount of drug is never in circulation at one time. The most well-known approach to nanoencapsulation of Dox is liposomal doxorubicin (Doxil®), FDA-approved in 1995 for Kaposi’s sarcoma. Despite proven clinical superiority and improved tolerability of liposomal doxorubicin, unique adverse events emerged with use (7). In an attempt to further refine Dox delivery, we utilized a sol-gel polymerization technique to create silane composite nanoparticles (8, 9). The platform was modified from a sol-gel-based protocol shown to successfully incorporate a range of therapeutic agents, including amphotericin (10) and sildenafil (11). These particles are formed from amorphous silicon oxide materials that polymerize into a highly structured porous lattice. The large surface area allows for greater drug loading as compared to liposomes (12), and the pore size distribution can be modified to alter the release rate of the encapsulated drug (13). Here we describe their synthesis and enhanced anti-cancer activity, relative to Dox, in cancer cell lines.

Table 1: Dox-np Synthesized with Varying Concentrations of methanol

0% MeOH nanoparticles

40% MeOH nanoparticles

60% MeOH nanoparticles

80% MeOH nanoparticles

100% MeOH nanoparticles

22 mL Tris (50mM)

12.4 mL Tris (50 mM)

7.6 mL Tris (50mM)

2.8 mL Tris (50 mM)

24 mL methanol

1.5 mL chitosan (5mg/ml)

9.6 mL methanol

14.4 mL methanol

19.2 mL methanol

1.5 mL chitosan

1.5 ml PEG 400

1.5 ml chitosan

1.5 ml chitosan

1.5 ml chitosan

1.5 ml PEG 400

2 mL adriamycin (2mg/ml)

1.5 mL PEG 400

1.5 mL PEG 400

1.5 mL PEG 400

2 mL adriamycin (2mg/ml)

3 mL TMOS

2 mL adriamycin (2mg/mL)

2 mL adriamycin (2mg/mL)

2 mL adriamycin (2mg/mL)

3 mL TMOS

0.6 mL HCl (1mM)

3 mL TMOS

3 mL TMOS

3 mL TMOS

0.6 mL HCl (1mM)

0.6 mL HCl (1mM)

0.6 mL HCl

0.6 mL HCl (1mM)

In the polymerization phase, the percent concentration of methanol was increased between different samples. 0%, 40%, 60%, 80% and 100% methanol content correspond to Dox-np (A0), (A40), (A60), (A80), and (A100), respectively. See methods for additional detail.

Materials and Methods

Synthesis of Dox nanoparticles (Dox-np)

Clinical grade doxorubicin hydrochloride solution (2 mg/mL) was obtained from Pfizer (New York, NY). A hydrogel/glass composite incorporating Dox as the active component was produced as follows: Tetramethyl orthosilicate (TMOS) was hydrolyzed by adding HCl, followed by 20-minute sonication in an ice water bath. The mixture was refrigerated at 4°C until monophasic. Subsequently, different ratios of Tris-buffered saline and methanol were combined with chitosan, polyethylene glycol, doxorubicin (2 mg/mL), and TMOS-HCl to induce sample polymerization overnight at 4°C (see Table 1 for quantities).

The percent concentration of methanol utilized in Dox-np synthesis was 0%, 40%, 60%, and 80% (represented as Dox-np A0, A40, A60, A80). The hydrogel was subsequently lyophilized at ~200 mTorr for 48–72 hours and the resultant powder processed in a ball mill for ten 30-minute cycles to achieve smaller size and more uniform distribution. Control nanoparticles (control-np) were synthesized identically but without the incorporation of Dox.

(See Supplemental Information for additional methods.)

Results

Dox-np Diameter Characterization

Different variants of Dox-np were synthesized by changing the percent concentration of methanol in the gel phase (Table 1). However, once lyophilized, methanol was removed from the final product, abrogating potential cytotoxicity. The change in size of Dox-np as a function of initial methanol content was determined using dynamic light scattering. Imaging of Dox-np with a scanning electron microscope exhibited a distinct spherical structure with an irregular surface morphology, as shown in Figure 1A. The most significant differences were observed with the Dox-np (A0) and (A80), with an average diameter of 118.6 and 103.4 nm, respectively (Figure 1B).

Dox-np Release Profile

The amount of encapsulated Dox based on release in DMSO was calculated to be 14.5±0.35 ug/ml. The effect of temperature on the release profile of Dox-np was assessed by measuring the spectrophotometric absorbance of a Dox-np solution over time at both 4 and 37°C. Temperatures were selected to simulate storage and in vivo conditions, respectively.

At 4 °C, Dox-np (A0) immediately released 24.6% of encapsulated Dox in solution (t=0 hours) with no further release over 48 hours. In comparison, Dox-np (A80) initially released 11.9% with no further release over time (Figure 1C).

Increasing the temperature to 37°C prompted the continuous release of Dox from the porous matrix in a controlled manner (Figure 1D). The curve for both nano-formulations was characterized by an initial accelerated release from t=0 until t=6 hours, followed by a gradual increase until steady state was reached at 24 hours. Dox-np (A80) had a slower rate of release compared to Dox-np (A0), with lower maximum release after 48 hours. At t=0 hours, Dox-np (A0) released 37.5%, increasing to 60% at 6 hours and reaching a steady state by 24 hours of 68.3%. In comparison, at t=0 Dox-np (A80) released 17.2%, increasing to 31.7% at 6 hours and reaching a steady state by 24 hours of 44.1% release. Since, Dox-np (A80) had a slower release curve compared to Dox-np (A60) and (A40 – not shown). Future cell-based experiments focused mainly on A0 and A80.

Overall, the addition of methanol during Dox-np synthesis correlated with both decreased particle size, and slower initial and maximal release of encapsulated Dox, relative to Dox-np synthesized in the absence of methanol (A0).

Anti-Tumor Efficacy of Dox-np In Cells

We evaluated in-cell activity using multiparametric dose-response modeling in a panel of genetically diverse cancer cell lines. This approach generates metrics to facilitate robust comparison of Dox versus Dox-np using area under the curve (AUC), EMax and EC50 as read outs of anti-cancer activity (14). Although EC50 is a commonly used metric of potency, it usually reflects a dose that suppresses proliferation. Dose-response curves for all cell lines indicated substantial variation between Dox, Dox-np (A0) and Dox-np (A80), as shown in Figure 2A.

Figure 1. Characterization of Dox-np Size and Effect of Temperature on Doxorubicin Release. (A) Representative scanning electron microscopy images of Dox-np particles. (B) Hydrodynamic diameter of nanoparticles assessed via dynamic light scattering revealed decreasing nanoparticle size with increasing methanol content. Scale bar = 200 nm, n=2. (C) At 4°C, minimal Dox was released in an initial burst at t = 0 hours with no further release over 48 hours. (D) At 37°C, Dox was released from the nanoparticle matrix with an acceleration from t=0-6 hours, followed by a gradual increase until steady state at 24 hours. Dox-np (A80) had a slower rate of release compared to Dox-np (A0), with lower maximum release after 48 hours. As depicted, theoretical maximum release was never achieved. Error bars denote χ ± sem. n=2.

Figure 2. Improved Dose-Response Relationship of Dox-np versus Doxorubicin In Triple-Negative Breast Cancer (TNBC). (A) Dose-response curves representing different variation in dose-response relationships. Patterns of dose-response are shown for Dox, Dox-np (A0) and Dox-np (A80). Each curve represents a dataset for one cell line. High variation was observed for doxorubicin and curves separate into 2 cohorts representing sensitive (S) and resistant (R) lines. This separation was also evident for Dox-np (A0). Dox-np (A80) gave a more uniform distribution. (B) Distribution of dose-response parameters AUC, EMax and EC50 for Dox-np (A80) relative to Dox, or Dox-np (A0) across 6 cancer cell lines. Values were computed from sigmoidal dose-response simulations and drawn as box and whisker plots showing median value (black horizontal line) with interquartile range (boxes). Bars extending to 1.5× the interquartile range indicate variance. Among all cell lines, EMax is most improved relative to Dox. (C) Distribution of dose-response parameters segregated according to Dox resistance or sensitivity. Resistant lines were mesenchymal TNBC’s (BL2 and M subtype). Dox-np (A80) had statistically significantly superior EMax relative to Dox in the resistant cohort (P < 0.0001; Wilcoxon signed rank test) with slightly increased AUC (P=NS) and statistically significantly decreased EC50 (P < 0.005; Wilcoxon signed rank test), consistent with improved anti-cancer activity overall. Outliers are shown as non-connected data points in the plots.

High variation was observed for Dox such that curves dichotomized according to steep versus shallow slope, demarcating sensitive (S) and resistant (R) cohorts, respectively. This trend was also apparent for Dox-np (A0); however, Dox-np (A80) dose-response curves were steeper in the resistant cohort specifically, generating a more uniform dose-response relationship across all cell lines (Figure 2A). Dose-response parameters computed from sigmoidal dose-response curves are summarized in Table 2. Median values across all cell lines (black horizontal line) are depicted as box and whisker plots showing interquartile range (boxes) and variance (bars extending to 1.5× the interquartile range (Figure 2B). This analysis indicated increased AUC and EMax for Dox-np (A0 and A80) relative to Dox, although only EMax reached statistical significance (P < 0.05; Wilcoxon signed rank test, Dox-np (A80) versus Dox). However, median EC50 increased for Dox-np, consistent with decreased sensitivity at doses associated with suppression of proliferation. Thus, improvements in dose-response parameters occurred at the high concentration range.

Table 2: Dose-Response Parameters for Dox versus Dox-np in a Panel of Cancer Cell Lines

EMax

AUC

EC50 (Log2)

Tumor Type

Cell Line

Dox

Dox-np (A80)

Dox

Dox-np (A80)

Dox

Dox-np (A80)

TNBC-BL2

Sum149PT

0.52

0.88

1.99

2.41

5.93

7.36

TNBC-M

Hs578T

0.68

0.88

2.01

2.11

8.45

7.66

TNBC-M

MDA-MB-157

0.50

0.80

1.92

3.02

9.63

6.92

TNBC-BL1

MDA-MB-468

0.89

0.95

5.07

4.33

3.89

5.13

NSCLC

A549

0.90

0.95

5.26

5.04

3.42

4.34

Ovarian

HEY

0.87

0.93

4.21

3.85

4.37

5.45

Dose-Response modeling enabled computation of metrics across 6 cancer cell lines, including Emax (a measure of efficacy), AUC (area under the dose-response curve) and EC50 (a measure of potency). Cell lines highlighted in grey represent the resistant cohort and are molecular subtypes of TNBC that are metastatic and have poor overall survival. Dox-np (A80) demonstrated dramatically superior EMax in these cell lines, relative to Dox.

TNBC – Triple negative breast cancer; BL1 – Basal-like 1; BL2 – Basal-like 2; M – Mesenchymal; NSCLC – Non-small cell lung carcinoma.

To facilitate a more nuanced evaluation of sensitive and resistant cohorts, dose-response parameters were recomputed for these 2 groups and box plots redrawn (Figure 2C). Resistant lines (SUM149PT, Hs578T and MDA-MB-157) were BL2 and mesenchymal (M) subtypes of TNBC that represent types of disease with aggressive, metastatic tumor biology (15). Dox-np (A80) had statistically significantly increased EMax relative to Dox in the resistant cohort (P < 0.001; Wilcoxon signed rank test) and slightly increased AUC and decreased EC50, consistent with improved anti-tumor activity overall. In the sensitive cohort, Dox had comparable ECMax as Dox-np, while AUC and EC50 values showed decreased effect of Dox-np relative to Dox. This lack of sensitization by Dox-np in sensitive cohorts is also shown in dose-response curves for individual cell lines (Figure S1). Thus, Dox-np has superior efficacy specifically in BL2 and M subtypes of TNBC in vitro.

Dox-np Has Superior Tumor Cell Kill in BL2 and M Subtypes of Breast Cancer

High-dose Dox-np (A80) caused enhanced tumor cell kill in chemoresistant TNBC cell lines (Figure 3). Dox was unable to elicit the same degree of tumor cell kill at equivalent concentrations (800nM). At lower doses (100 nM), the difference in survival between Dox and Dox-np was not remarkable, consistent with other data indicating that the superiority of Dox-np is limited to high concentrations.