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Peripherally Cross-Linking the Shell of Core-Shell Polymer Micelles Decreases Premature Release of Physically Loaded Combretastatin A4 in Whole Blood and Increases its Mean Residence Time and Subsequent Potency Against Primary Murine Breast Tumors After IV Administration

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ABSTRACT

Purpose

Determine the feasibility and potential benefit of peripherally cross-linking the shell of core-shell polymer micelles on the premature release of physically loaded hydrophobic drug in whole blood and subsequent potency against solid tumors.

Methods

Individual Pluronic F127 polymer micelles (F127 PM) peripherally cross-linked with ethylenediamine at 76% of total PEO blocks (X-F127 PM) were physically loaded with combretastatin A4 (CA4) by the solid dispersion method and compared to CA4 physically loaded in uncross-linked F127 PM, CA4 in DMSO in vitro, or water-soluble CA4 phosphate (CA4P) in vivo.

Results

X-F127 PM had similar CA4 loading and aqueous solubility as F127 PM up to 10 mg CA4 / mL at 22.9 wt% and did not aggregate in PBS or 90% (v/v) human serum at 37°C for at least 24 h. In contrast, X-F127 PM decreased the unbound fraction of CA4 in whole blood (fu) and increased the mean plasma residence time and subsequent potency of CA4 against the vascular function and growth of primary murine 4T1 breast tumors over CA4 in F127 PM and water-soluble CA4P after IV administration.

Conclusions

Given that decreasing the fu is an indication of decreased drug release, peripherally cross-linking the shell of core-shell polymer micelles may be a simple approach to decrease premature release of physically loaded hydrophobic drug in the blood and increase subsequent potency in solid tumors.

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Abbreviations

4 T1:

Breast tumor epithelial cells from BALB/c mice

4 T1-Luc:

4 T1 cells that stably express luciferase

AFM:

Atomic force microscopy

CA4:

Combretastatin A4

CA4P:

Combretastatin A4 Phosphate / Fosbretabulin disodium

DLS:

Dynamic light scattering

DMSO:

Dimethyl sulfoxide

DSC:

N,N’-Disuccinimidyl carbonate

ECIS:

Electric cell-substrate impedance sensing

ED:

Ethylenediamine

F127:

Pluronic F127 (Poloxamer 407)

F127 PM:

Pluronic F127 polymer micelles

fu :

Unbound fraction of drug in whole blood

HUVEC:

Human umbilical vein endothelial cells

IVIS:

In vivo imaging system

NHS-F127:

DSC-activated Pluronic F127

NHS-F127 PM:

DSC-activated Pluronic F127 polymer micelles

X-F127 PM:

Pluronic F127 polymer micelle shell cross-linked with ED at 76%

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ACKNOWLEDGEMENTS AND DISCLOSURES

This work was supported by NIH COBRE grant 2P20GM103480-06 (Nebraska Center for Nanomedicine) (RRW, RKS, JAV), NIH 1U54CA163120-01 grant (RKS), and UNMC Predoctoral Fellowships (SPRB, ST, VVA). The Nanoimaging Core Facility was supported by the NIH (SIG program), the UNMC Program of Excellence (POE), and the Nebraska Research Initiative (NRI). The Authors would also like to acknowledge Todd A. Wyatt, PhD and the VA Nebraska-Western Iowa Health Care System Research Service for providing access to and assistance with the Electric Cell Impedance Sensing apparatus.

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Authors and Affiliations

  1. Center for Drug Delivery and Nanomedicine, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, Nebraska, 68198-6025, USA

    Rakesh K. Singh & Joseph A. Vetro

  2. Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, Nebraska, 68198-6025, USA

    Rajesh R. Wakaskar, Sai Praneeth R. Bathena, Shailendra B. Tallapaka, Nagsen Gautam, Rhishikesh Thakare, Stephen M. Curran, Yuxiang Dong & Joseph A. Vetro

  3. Nanotechnology Characterization Laboratory, Cancer Research Technology Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland, 21702, USA

    Vishakha V. Ambardekar

  4. Department of Pulmonary, Critical Care, Sleep, & Allergy, College of Medicine, University of Nebraska Medical Center, 981050 Nebraska Medical Center, Omaha, Nebraska, 68198-1050, USA

    Samantha M. Simet

  5. Department of Pathology and Microbiology, University of Nebraska Medical Center, 985900 Nebraska Medical Center, Omaha, Nebraska, 68198-5900, USA

    Rakesh K. Singh

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  1. Rajesh R. Wakaskar
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Corresponding author

Correspondence to Joseph A. Vetro.

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Fig. S1

Key peak assignments for 1H-NMR spectra of NHS-activated F127 alone or F127 polymer micelles with exclusively monovalent or divalent (cross-linked) conjugation of ethylenediamine (ED). (A) NHS-activated F127 (NHS-F127) or polymer micelles of NHS-F127 reacted with (B) a 5/1 molar ratio of ED/NHS-F127 that produced only monovalent ED conjugation to PEO blocks of F127 PM (Monovalent ED) or (C) a 1/1 molar ratio of ED/NHS-F127 that produced only divalent ED conjugation (i.e., cross-linking) between PEO blocks of F127 PM (Divalent ED) were compared by 1H-NMR (500 MHz, CDCl3, 1 mg/mL). NHS-F127: δ 1.01–1.26 (m, 195H, peak 5), 2.85 (s, 8H, peak 2), 3.25–3.82 (m, ca. 991H, peak 3), 4.46 (t, J = 5.2 Hz, 4H, peak 1); F127 PM with monovalent and/or divalent ED conjugation: δ 1.01–1.26 (m, 195H, peak 5), 3.12 (brs, peak 4), 3.25–3.82 (m, ca. 991H, peak 3), 4.22 (brs, peak 1A). The proton peak at 4.22 ppm (peak 1A) represents both monovalent and divalent ED conjugation and the proton peak at 3.12 ppm (peak 4) represents monovalent ED conjugation alone. (GIF 52.3 kb)

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Fig. S2

Effect of peripherally cross-linking the shell of F127 PM on the pharmacokinetics of physically loaded CA4 after IV administration in 4 T1 tumor-bearing mice. Uncross-linked F127 polymer micelles (F127 PM) or F127 PM individually cross-linked with ED at 76% of total PEO blocks (X-F127 PM) were loaded with CA4 at 22.9 wt% as described in Fig. 5. 4 T1 cells stably expressing luciferase (4 T1-Luc) were injected SQ into the mammary fat pad of female BALB/c mice and grown until ~100 mm3 before treatment. Water-soluble CA4P (closed circles) or CA4 loaded in F127 PM (closed squares) or X-F127 PM (cross-hatched squares) was then injected IV (1 mg / kg) and the average concentration of CA4 in plasma ± SEM (n = 6 mice / time point) was determined by LC-MS/MS. (GIF 39.4 kb)

High Resolution Image (TIFF 98.9 kb)

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Wakaskar, R.R., Bathena, S.P.R., Tallapaka, S.B. et al. Peripherally Cross-Linking the Shell of Core-Shell Polymer Micelles Decreases Premature Release of Physically Loaded Combretastatin A4 in Whole Blood and Increases its Mean Residence Time and Subsequent Potency Against Primary Murine Breast Tumors After IV Administration. Pharm Res 32, 1028–1044 (2015). https://doi.org/10.1007/s11095-014-1515-z

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