Ion-containing Poly(aminophosphonate)- based Nanocarriers for Simultaneous Magnetic Resonance Imaging and Drug Delivery

Transcription

1 Polymers in Medicine and Biology October, 2013 Ion-containing Poly(aminophosphonate)- based Nanocarriers for Simultaneous Magnetic Resonance Imaging and Drug Delivery Nikorn Pothayee and A. P. Koretsky, Laboratory for Functional and Molecular Imaging, NINDS, NIH, Bethesda, MD and Nipon Pothayee and J. S. Riffle Macromolecules & Interfaces Institute, Virginia Tech Blacksburg, VA

2 Objective Design complexes that can carry and release cancer therapeutics with simultaneous MRI positive imaging Potentially track biodistribution of drug complexes in vivo

3 Outline for today Poly(ammonium bisphosphonate) graft copolymers and MaGIC manganese complexes Strong binding of polymer to manganese little to no release in PBS and no displacement by calcium ions High relaxivities and potentially good image contrast Stable colloidal stability Early toxicity tests are promising Co-encapsulation of anticancer drugs and manganese ions into MaGICs for potential simultaneous imaging and delivery Good uptake of cancer drugs with release rates dependent on the drug structure Good anticancer efficacies against breast and brain cancer cell lines

4 Magnetic Resonance Imaging (MRI) as a Tool for High Resolution In Vivo Imaging Contrast Agent-assisted MRI Loss signal/dark image = negative contrast Facilitates decay of signal due to T 2 -relaxation liver liver Pre-contrast Post-contrast Gain signal/bright image = posititive contrast Facilitates recovery of signal due to T 1 - relaxation liver liver 4 Pre-contrast Post-contrast

5 Manganese-based Contrast Agents as Alternatives to Gadolinium Mn spin number 5/2 Labile water exchange Natural cellular components Approved Mn contrast agents Mn-DPDP (dipyridoxyl diphosphate) Mn-DPDP (Teslascan) Although no relation between Mn and nephrogenic systemic fibrosis has been found so far, the free form of Mn 2+ is known to pose some neurotoxicity. However, this issue can be solved by chelation of free Mn 2+ with a chelating agent or polymeric nanomaterial to form a stable complex.

6 Graft Copolymers in this Study Carboxyl 52:48 wt:wt PAA:PEO Propyl 60:40 wt:wt PABP:PEO Hexyl 59:41 wt:wt PABP:PEO Molecular weight of PEO = 5000 g/mole

7 Synthesis of β-aminobisphosphonate monomers

8 Synthesis of Polyaminobisphosphonate-g-PEO M n = 5K

9 Synthesis of MaGICs Mn 2+ DI water ph 7.4 Stirred for 24 h Dialyzed for 2 days Poly(ammonium bisphosphonate)-g-peo MaGICs Complex Moles of P/Mn or C/Mn Intensity average diameter (nm) Zeta Potential (mv) Carboxyl MaGICs Propyl MaGICs Hexyl MaGICs Carboxyl MaGICs Propyl MaGICs Hexyl MaGICs

10 Dispersions of Phosphonate MaGICs are Stable in PBS, ph 7.4 at RT but Carboxyl MAGICs are Not The MaGICs had excellent colloidal stability in phosphate buffered saline (PBS) for up to 24 h. This suggests that they will be sufficiently stable under physiological conditions to be suitable contrast agent.

11 Mn in the Phosphonate MaGICs does not Release in PBS (ph 7.4 at 37 C) but it is Unstable in Carboxyl MAGICs % Accumulated release % 45 % 7 % 0 % Time (h) Propyl-MaGICs 3.3 Hexyl-MaGICs 3.3 Carboxyl MaGICs 3.3 (control) MnCl2 At ph 7.4, Propyl and Hexyl MaGICs significantly slow down the release of Mn reaching ~ 0-7% in 24 h compared to Carboxyl MaGICs that release ~45 % of Mn within 24 h and free Mn that fully diffused through the dialysis membrane within 9 hours

12 NMR Relaxivities of MaGICs Measured at 1.4 Tesla, 37 C Complex Intensity ave Moles of P/Mn or C/Mn r 1 r 2 /r diameter 1 (nm) MnCl Carboxyl MaGICs Propyl MaGICs Hexyl MaGICs Carboxyl MaGICs Propyl MaGICs Hexyl MaGICs Mn-DPDP

13 T 1 - & T 2 -weighted Phantom Images (7 T, 25 C) T1-weighted T2-weighted μm metal ion GdDTPA MnDPDP Propyl MaGICS 2.0 Propyl MaGICs 3.3 Hexyl MaGICS 2.0 Hexyl MaGICS 3.3

14 Pre-injection In vivo MR imaging of MaGICs 15 μmol/kg MaGICs-CBPt 7.7 i.v. 40 min L S K 15 μmol/kg GdDTPA i.v. Bruker Pharmascan 7T, 16 cm bore Pre-injection 40 min GdDTPA MRI in C57BL6 mice at 7 T revealed excellent contrast signal enhancement (30-40%) exerted by the MaGICs at a dose fold lower than clinical dosage of Gd-based agents L = liver, S = spleen, K = kidney 14

15 Ca 2+ does not displace the Mn and doesn t affect the relaxivities of MaGICs R Measured at 1.4 Tesla, 37 C r 2 r mm (Mn) MnCl 2 Hexyl MaGICs (3.3) Hexyl MaGICs (3.3) mm Ca Since Ca 2+ is the most plentiful mineral found in the human body, stability of the MaGICs against Ca 2+ displacement is important! R mm (Mn)

16 MTS assay of MaGICs and Polymers in Murine Hepatocyte AML-12 Cells Free polymer Mn 2+ -Complexes Prop yl Hexyl MnCl2 P-MaGICs % proliferation % proliferation H-MaGICs Polymer concentration (ug/ml) Mn concentration (um) *MaGICs appear to mitigate cellular toxicity of Mn 2+ in AML-12 cells

17 Encapsulation of anticancer drugs into MaGICs

18 Doxorubicin-loaded MaGICs have comparable anti-proliferative effects relative to free doxurubicin Cytotoxic effect of MaGICs-DOX 14.5 and free DOX against MCF-7 breast cancer cell using an MTT assay

19 Proposed structures of Pt drug-loaded MaGICs MaGICs-Cisplatin MaGICs-Carboplatin fast ligand exchange reaction formed two coordination bonds slow ligand exchange reaction formed only one coordination bond strong interactions: electrostatic interactions + chelation 19

20 Release of Carboplatin in PBS is Significantly Faster than Cisplatin, ph 7.4 at 37 C 120 Carboplatin 120 Cisplatin % Accumulated release % Free CBPt MaGICs-CBPt 13.0 MaGICs-CBPt 7.7 MaGICs-CBPt % 22 % 11 % % Accumulated release Time (h) 100 % 13 % 3 % 0 % Time (h) Free CPt MaGICs-CPt 5.0 MaGICs-CPt 9.7 MaGICs-CPt 16.0 The release rate of carboplatin from MaGICs was found to be faster than that of cisplatin but slower than doxorubicin

21 Characteristics of MaGICs loaded with Carboplatin Diameter (nm) Zeta Potential (mv) Sample Pt (wt %) Intensity average diameter (nm) PDI Zeta potential (mv) MaGICs MaGICs-CBPt MaGICs-CBPt MaGICs-CBPt * By ICP-AES

22 Carboplatin-loaded MaGICs enhance anti-proliferative effects relative to free carboplatin by ~30-fold, whereas Cisplatin-loaded MaGICs are not effective Carboplatin Cisplatin Cytotoxic effect of MaGICs-CBPt 13 and free CBPt against MCF-7 breast cancer cell using MTT assay

23 In Vitro Screening on U251 Glioblastoma Cells Showed EnhancedAnti-proliferative Activities 120 % Proliferation Pt (um) cisplatin carboplatin MaGICs-CBPt 4.7 MaGICs-CBPt 7.7 MaGICs-CBPt 13 IC50 (μm) cisplatin 7.0 carboplatin 50 MaGICs-CBPt MaGICs-CBPt MaGICs-CBPt estimated from Graphpad software *Measured by MTT assay after continuous exposure of 48 h 23

24 Increase in anti-proliferative activity is not due to enhanced intracellular uptake of Pt 120 Intracellular Pt accumulation after 4 h incubation % Proliferation MaGICs-CBPt 13.0 Carboplatin Pt pg/cell Pt (um) 4 h exposure to complexes and free drug followed by 44 h of incubation 0 Carboplatin MaGICs-CBPt 13.0 * Measured by ICP of total 1,000,000 cell lysates

25 Enhanced anti-proliferative activities are not likely caused by cytotoxicity from MaGICs carriers 120 % Proliferation MaGICs-CBPt 4.7 Corresponding amount of polymeric carriers Pt (um) 25

26 Can MaGICs Overcome Drug Resistance? OVCAR8-Cisplatin IC50 (μm) % Proliferation Pt (um) NCI/ADR-RES-Cisplatin OVCAR8-Cisplatin NCI/ADR-RES-MaGICs- CBPt4.7 NCI/ADR-RES-Cisplatin NCI/ADR-RES-MaGICs- CBPt13.0 Cisplatin OVCAR8 Cisplatin NCI/ADR-RES MaGICs-CBPt 4.7 MaGICs-CBPt NCI/ADR-RES that express high levels of MDR-1 (PgP) also exhibit cross-resistance to cisplatin ~ 10X relative to its parental cell line OVCAR8 MaGICs-CBPts have superior antiproliferative activities than cisplatin in ADR-RES cells Among the complexes, MaGICs-CBPt 4.7 has the highest activity. Conversely, in vitro release profiles suggest that release of Pt species is slowest relative to other complexes. Is release of Pt species necessary to exert killing activity? 26

27 Summary Complexation of Mn with poly(ammonium bisphosphonate)-g-peo significantly increases r 1 and decreases the r 2 /r 1, thus improving the contrast Propyl and Hexyl MaGICs essentially eliminate release of the Mn compared to Carboxylate complexes Physiological concentration of Ca 2+ does not displace the Mn from the complexes Anticancer drugs were successfully encapsulated into MaGICs and their release depends on drug structure and concentration These manganese nanocarriers have excellent relaxometric properties together with anticancer activity against cancer cells. Carboplatin-loaded MaGICs enhanced the anti-proliferative effect of MCF-7 breast cancer cells relative to free carboplatin by almost 30-fold.

28 Acknowledgements NIND/NIH Intramural Research Program Developmental Therapeutic Program, DCTC cell/tumor repository, National Cancer Institute 28